CN113475437B - Method for breeding eggs of whitmania pigra - Google Patents
Method for breeding eggs of whitmania pigra Download PDFInfo
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- CN113475437B CN113475437B CN202110893265.9A CN202110893265A CN113475437B CN 113475437 B CN113475437 B CN 113475437B CN 202110893265 A CN202110893265 A CN 202110893265A CN 113475437 B CN113475437 B CN 113475437B
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- 238000009395 breeding Methods 0.000 title claims abstract description 82
- 230000001488 breeding effect Effects 0.000 title claims abstract description 78
- 235000013601 eggs Nutrition 0.000 title claims abstract description 52
- 241000258623 Whitmania pigra Species 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 16
- 241000545744 Hirudinea Species 0.000 claims abstract description 76
- 239000002689 soil Substances 0.000 claims abstract description 38
- 239000002131 composite material Substances 0.000 claims abstract description 21
- 230000013011 mating Effects 0.000 claims abstract description 17
- 239000012528 membrane Substances 0.000 claims abstract description 16
- 238000011534 incubation Methods 0.000 claims abstract description 13
- 239000004568 cement Substances 0.000 claims abstract description 12
- 230000004720 fertilization Effects 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 74
- 229920001661 Chitosan Polymers 0.000 claims description 39
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 33
- 108010010803 Gelatin Proteins 0.000 claims description 30
- 229920000159 gelatin Polymers 0.000 claims description 30
- 239000008273 gelatin Substances 0.000 claims description 30
- 235000019322 gelatine Nutrition 0.000 claims description 30
- 235000011852 gelatine desserts Nutrition 0.000 claims description 30
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 claims description 29
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 238000003756 stirring Methods 0.000 claims description 26
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000000843 powder Substances 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 20
- 229910021641 deionized water Inorganic materials 0.000 claims description 20
- 239000011259 mixed solution Substances 0.000 claims description 20
- 239000002244 precipitate Substances 0.000 claims description 20
- 238000001035 drying Methods 0.000 claims description 16
- 238000010438 heat treatment Methods 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- 238000004519 manufacturing process Methods 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 13
- 230000000249 desinfective effect Effects 0.000 claims description 12
- 230000012447 hatching Effects 0.000 claims description 12
- 239000004005 microsphere Substances 0.000 claims description 11
- 239000002105 nanoparticle Substances 0.000 claims description 11
- ODINCKMPIJJUCX-UHFFFAOYSA-N Calcium oxide Chemical compound [Ca]=O ODINCKMPIJJUCX-UHFFFAOYSA-N 0.000 claims description 10
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 10
- 239000007787 solid Substances 0.000 claims description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 6
- FPCJKVGGYOAWIZ-UHFFFAOYSA-N butan-1-ol;titanium Chemical compound [Ti].CCCCO.CCCCO.CCCCO.CCCCO FPCJKVGGYOAWIZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003995 emulsifying agent Substances 0.000 claims description 6
- 235000019441 ethanol Nutrition 0.000 claims description 6
- 210000003608 fece Anatomy 0.000 claims description 6
- 239000010871 livestock manure Substances 0.000 claims description 6
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims description 5
- 239000000292 calcium oxide Substances 0.000 claims description 5
- 235000012255 calcium oxide Nutrition 0.000 claims description 5
- 239000000460 chlorine Substances 0.000 claims description 5
- 229910052801 chlorine Inorganic materials 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 239000010902 straw Substances 0.000 claims description 5
- 230000008961 swelling Effects 0.000 claims description 5
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 4
- 230000001954 sterilising effect Effects 0.000 claims description 4
- 238000001132 ultrasonic dispersion Methods 0.000 claims description 2
- 230000004083 survival effect Effects 0.000 abstract description 9
- 229910001385 heavy metal Inorganic materials 0.000 abstract description 8
- 229910052793 cadmium Inorganic materials 0.000 abstract description 3
- 229910052802 copper Inorganic materials 0.000 abstract description 3
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 3
- 210000004392 genitalia Anatomy 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 241000607479 Yersinia pestis Species 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000007885 magnetic separation Methods 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 210000003899 penis Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000000447 pesticide residue Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 241000237902 Hirudo medicinalis Species 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 241000243818 Annelida Species 0.000 description 1
- 241000719837 Anoectochilus Species 0.000 description 1
- 241000360040 Bdellidae Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 231100001240 inorganic pollutant Toxicity 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
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- 231100000956 nontoxicity Toxicity 0.000 description 1
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
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- 238000001179 sorption measurement Methods 0.000 description 1
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Classifications
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- 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
- A01K61/40—Culture of aquatic animals of annelids, e.g. lugworms or Eunice
-
- 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
- A01K61/10—Culture of aquatic animals of fish
- A01K61/17—Hatching, e.g. incubators
-
- 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
- A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
- A01K63/003—Aquaria; Terraria
-
- 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
- A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
- A01K63/003—Aquaria; Terraria
- A01K63/006—Accessories for aquaria or terraria
-
- 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
- A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
- A01K63/04—Arrangements for treating water specially adapted to receptacles for live fish
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Marine Sciences & Fisheries (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Zoology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an egg breeding method for whitmania pigra, and belongs to the technical field of aquatic breeding. The method for breeding the eggs of the whitmania pigra in the invention comprises the following steps: the method comprises the following steps of preparation of a breeding place, leech selection, leech disinfection, mating fertilization and egg cocoon incubation, wherein the breeding place is convenient for the incubation and growth of the whitmania pigra, the breeding success rate of the whitmania pigra can be remarkably improved, an incubator can provide a reliable growth environment for the whitmania pigra, the breeding efficiency of the whitmania pigra is improved, a modified composite membrane is laid in a cement breeding pool and used for adsorbing heavy metals Cu and Cd in soil, the safety of the soil is guaranteed, the egg cocoon incubation survival rate is promoted to reach 92%, the quality of the whitmania pigra fry is improved, the breeding risk is reduced, and great economic benefits are achieved for the healthy development of the whitmania pigra artificial breeding industry.
Description
Technical Field
The invention belongs to the technical field of aquaculture and relates to an egg breeding method for whitmania pigra.
Background
Whitmania pigra Whitman belongs to Annelida, bdellidae, arundinalales, whitmaniaceae and Anoectochilus, is commonly called leech, is one of three medicinal leech breeding varieties in China, and the dry product thereof is a traditional Chinese medicinal material with special effect in China. The medicinal material market has huge demand for medicinal leeches, and because the wild resources are almost exhausted due to environmental pollution and artificial over-fishing, the wild leech fishing yield cannot meet the market demand. The artificial breeding work of the whitmania pigra can be roughly divided into the following steps: the method comprises the following steps of leech seed introduction, egg cocoon breeding, egg cocoon collection and incubation, fry cultivation and daily management. Breeding leech egg cocoons in a ridge built outdoors. The substrate adopted by the leech breeding and egg cocoon hatching platform is mainly farmland soil. The conventional seedling raising method has many disadvantages, such as: the problems of moisture retention, air permeability, softness, insect damage and the like of the cocoon production and incubation platform built by farmland soil are difficult to control; the workload is large, and time and labor are consumed; the method is easily influenced by weather conditions, the emergence rate is low, and the production stability of seedlings is poor; more importantly, the farmland soil also has bacteria, microorganisms, pesticide residues and heavy metal residues which can threaten the survival of the leech seeds, so that the development of an egg breeding method of the whitmania pigra whitman is needed, and the safety of egg breeding and the quality of offspring seeds are ensured.
Disclosure of Invention
The invention aims to provide an egg culturing method for whitmania pigra, which is used for avoiding threat to the survival of whitmania pigra caused by bacteria, microorganisms, pesticide residues and heavy metal residues existing in soil, ensuring the safety of an egg culturing environment, not only disinfecting and sterilizing a cement breeding pool, but also paving a modified composite membrane in the cement breeding pool for adsorbing heavy metals of Cu and Cd in the soil, ensuring the safety of the soil and promoting the survival rate of hatching of egg cocoons to be as high as 92 percent.
The purpose of the invention can be realized by the following technical scheme:
a method for breeding eggs of whitmania pigra, which comprises the following steps:
step A1, preparation of a breeding place: building a greenhouse, building cement breeding pools in the greenhouse, wherein the width of each breeding pool is 0.6-0.8m, the height of each breeding pool is 1-1.2m, the distance between every two adjacent breeding pools is 1.3-1.5m, disinfecting the breeding pools by strong chlorine, airing for 7-10 days, laying the modified composite membrane in the breeding pools, adding loess as a production bed, the depth of the loess is 0.6-0.7m, and adding fermented farmyard manure;
step A2, leech seed selection: the leech seed is formed by selecting leech which is bright in color, strong, thick, lively and movable and can be quickly shrunk into a ball after being touched by hand;
step A3, disinfecting leeches: disinfecting the leech seeds for 20min, then lightly putting the leech seeds on a production bed of a breeding pond, and covering with straws;
step A4, mating and fertilization: placing leeches, enabling the head ends of the leeches to be opposite in direction, enabling the ventral surfaces of the leeches to lean together, enabling respective male organs to just face the female genital orifice of the other party, then inserting the thin linear penis extending out of the male into the female genital orifice of the other party, enabling the mating time to last for 2-3h, and ensuring the quiet surrounding environment during mating;
step A5, hatching the egg cocoons: after 30 days, the egg cocoons of the leeches are pulled out from the obstetric table, 2-3 egg cocoons of each leech are transferred into an incubator, a layer of incubation soil with the thickness of 1.5cm is paved at the bottom of the incubator, pointed egg cocoons are placed upwards on the incubation soil, a layer of incubation soil with the thickness of 1.5cm is covered on the pointed egg cocoons, the young leeches begin to grow after 20 days of incubation, and the young leeches are placed into a fine culture pond.
Further, in the step A1, quicklime is used for removing pests and harmful bacteria in soil, so that the breeding place of leeches is prevented from being damaged, and broken egg cocoons are avoided, and the use amount is 60-80 kg/mu.
Further, the temperature in the incubator in the step A5 is 20-27 ℃, the relative humidity of air is 75%, and the humidity of the hatching soil is 40%.
Further, the modified composite membrane is prepared by the following steps:
step S1, dispersing Fe3O4 in an ethanol solution with the mass fraction of 95%, adding ammonia water with the mass fraction of 28%, ultrasonically dispersing for 20-25min, placing the mixture in a water bath kettle at 45 ℃ for stirring and reacting for 1-2h to obtain a mixed solution a, dissolving butyl titanate in absolute ethyl alcohol to obtain a mixed solution b, dripping the mixed solution b into the mixed solution a, carrying out mixed reaction for 24-26h, carrying out magnetic separation to obtain solid particles, and washing the solid particles for 2-3 times by deionized water and absolute ethyl alcohol respectively to obtain Fe3O4-TiO2 microspheres;
s2, adding Fe3O4-TiO2 microspheres into a reaction kettle, adding 1mol/L sodium hydroxide solution, heating to 140 ℃, reacting for 2-3h, centrifuging to obtain a precipitate, washing the precipitate for 2-3 times by deionized water and absolute ethyl alcohol respectively, and drying for 9-12h at 40 ℃ to obtain Fe3O4-TiO2 nanoparticles;
s3, dissolving chitosan in an acetic acid solution with the mass fraction of 2%, stirring for reaction for 12 hours, adding Fe3O4-TiO2 nanoparticles, span-80 and OP-10 emulsifier, uniformly mixing, adding a glutaraldehyde solution with the mass fraction of 25%, heating to 40-45 ℃, carrying out water bath reaction for 2-3 hours, adjusting the pH to 9-10 by using 1mol/L sodium hydroxide solution, centrifuging to obtain a precipitate, washing the precipitate with deionized water and absolute ethyl alcohol for 2-3 times respectively, and drying at 60-70 ℃ to constant weight to obtain Fe3O4-TiO 2-chitosan powder;
s4, adding Fe3O4-TiO 2-chitosan powder into an acetic acid solution with the volume fraction of 1%, swelling for 36-48h at room temperature, and stirring at the rotating speed of 300-320rpm until the solution is dissolved to obtain a Fe3O4-TiO 2-chitosan solution;
step S5, adding gelatin powder and deionized water into a three-neck flask, heating to 60 ℃, and magnetically stirring until the gelatin powder and the deionized water are dissolved to obtain a gelatin solution;
and S6, uniformly mixing the Fe3O4-TiO 2-chitosan solution and the gelatin solution, adding glycerol, uniformly stirring, paving the film, drying in a 50 ℃ oven for 40-48h, and removing the film to obtain the modified composite film.
Further, the dosage ratio of Fe3O4, ethanol solution, ammonia water, butyl titanate and absolute ethyl alcohol in the step S1 is 60.5-62.5mg:40-45mL:0.3-0.4mL:1.5-2mL:20-25mL.
Further, the dosage ratio of the Fe3O4-TiO2 microspheres and the sodium hydroxide solution in the step S2 is 60.2-61.5mg:40-42mL.
Further, the dosage ratio of the chitosan, the acetic acid solution, the Fe3O4-TiO2 nano particles, the span-80, the OP-10 emulsifier and the glutaraldehyde solution in the step S3 is 2.1-2.3g:100-103mL:0.4-0.42g:20-20.5mL:120-122mL:2.1-2.4mL.
Further, the dosage ratio of the Fe3O4-TiO 2-chitosan powder and the acetic acid solution in the step S4 is 14.2-14.6g:700-705mL.
Further, the dosage ratio of the gelatin powder and the deionized water in the step S5 is 6.3-6.5g:100-105mL.
Further, the mass ratio of the Fe3O4-TiO 2-chitosan solution to the gelatin solution in the step S6 is 1:0.45 percent, and the dosage of the glycerol is 15 percent of the total mass of the Fe3O4-TiO 2-chitosan solution and the gelatin solution.
The invention has the beneficial effects that: the invention aims to provide an egg breeding method of whitmania pigra, which comprises the steps of firstly building a cement breeding pool as a breeding place in a greenhouse, paving a modified composite membrane in the cement breeding pool, then placing loess as a production bed, covering fermented farmyard manure on the production bed, selecting robust whitmania pigra, sterilizing and placing on the production bed for mating fertilization, then transferring egg cocoons of the whitmania pigra to an incubator, and setting a proper incubation condition for incubation. In addition, in consideration of the safety of an egg breeding environment, the cement breeding pond is disinfected and sterilized, bacteria, microorganisms, pesticide residues and heavy metal residues in soil can also threaten the survival of leeches, therefore, a modified composite membrane is laid in the cement breeding pond and used for adsorbing heavy metals of Cu and Cd in the soil, the modified composite membrane mainly comprises Fe3O4, tiO2, chitosan and gelatin, a semiconductor material TiO2 has chemical inertness and nontoxicity and unique capability of removing organic/inorganic pollutants in wastewater and air, and has the characteristics of high activity, good stability and low cost, a magnetic adsorbent Fe3O4 has a chemical active surface and a large surface area, can extract heavy metal ions, the chitosan has high contents of amino and hydroxyl groups which have isolated charges, and strong chemical adsorption effect on the heavy metals, and the three cooperate with each other, further ensures the safety of soil incubation, promotes the survival rate of egg cocoons to 92%, promotes the quality of wide-body leeches, reduces the breeding risk, and has great economic benefit on the healthy development of the wide-body leech fry breeding industry.
Detailed Description
The technical solutions of the present invention will be described below clearly and completely in conjunction with the embodiments, and it is obvious 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.
Example 1
The modified composite membrane is prepared by the following steps:
step S1, dispersing 60.5mgFe 3O4 in 40mL of ethanol solution with the mass fraction of 95%, adding 0.3mL of ammonia water with the mass fraction of 28%, ultrasonically dispersing for 20min, placing the mixture in a 45 ℃ water bath kettle, stirring and reacting for 1h to obtain mixed solution a, and dissolving 1.5mL of butyl titanate in 20mL of absolute ethanol to obtain mixed solution b; dropping the mixed solution b into the mixed solution a, carrying out mixing reaction for 24 hours, then carrying out magnetic separation to obtain solid particles, and washing the solid particles for 2 times respectively by using deionized water and absolute ethyl alcohol to obtain Fe3O4-TiO2 microspheres;
s2, adding 60.2mgFe 3O4-TiO2 microspheres into a reaction kettle, adding 40mL of 1mol/L sodium hydroxide solution, heating to 140 ℃, reacting for 2 hours, centrifuging to obtain a precipitate, washing the precipitate for 2 times by deionized water and absolute ethyl alcohol respectively, and drying for 9 hours at 40 ℃ to obtain Fe3O4-TiO2 nanoparticles;
s3, dissolving 2.1g of chitosan in 100mL of acetic acid solution with the mass fraction of 2%, stirring for reaction for 12h, adding 0.4g of Fe3O4-TiO2 nanoparticles, 20mLspan-80 and 120mLOP-10 emulsifier, uniformly mixing, adding 2.1mL of glutaraldehyde solution with the mass fraction of 25%, heating to 40 ℃ for water bath reaction for 2h, adjusting the pH to 9 by using 1mol/L of sodium hydroxide solution, centrifuging to obtain precipitates, washing the precipitates for 2 times by using deionized water and absolute ethyl alcohol respectively, and drying at 60 ℃ to constant weight to obtain Fe3O4-TiO 2-chitosan powder;
s4, adding 14.2gFe 3O4-TiO 2-chitosan powder into 700mL of acetic acid solution with volume fraction of 1%, swelling for 36h at room temperature, and stirring at the rotating speed of 300rpm until the chitosan is dissolved to obtain Fe3O4-TiO 2-chitosan solution;
step S5, adding 6.3g of gelatin powder and 100mL of deionized water into a three-neck flask, heating to 60 ℃, and magnetically stirring until the gelatin powder is dissolved to obtain a gelatin solution;
step S6, uniformly mixing the Fe3O4-TiO 2-chitosan solution and the gelatin solution, adding glycerol, uniformly stirring, paving a film, drying in a 50 ℃ drying oven for 40 hours, and removing the film to obtain a modified composite film, wherein the mass ratio of the Fe3O4-TiO 2-chitosan solution to the gelatin solution is 1:0.45 percent, and the dosage of the glycerol is 15 percent of the total mass of the Fe3O4-TiO 2-chitosan solution and the gelatin solution.
Example 2
The modified composite membrane is prepared by the following steps:
s1, dispersing 61.5mgFe 3O4 in 43mL of ethanol solution with the mass fraction of 95%, adding 0.35mL of ammonia water with the mass fraction of 28%, ultrasonically dispersing for 20min, placing the mixture in a water bath kettle at 45 ℃, stirring and reacting for 1h to obtain mixed solution a, and dissolving 1.7mL of butyl titanate in 23mL of absolute ethanol to obtain mixed solution b; dripping the mixed solution b into the mixed solution a, performing mixing reaction for 25 hours, then performing magnetic separation to obtain solid particles, and washing the solid particles with deionized water and absolute ethyl alcohol for 2 times respectively to obtain Fe3O4-TiO2 microspheres;
step S2, adding 60.8mgFe 3O4-TiO2 microspheres into a reaction kettle, adding 41mL of 1mol/L sodium hydroxide solution, heating to 140 ℃, reacting for 2 hours, centrifuging to obtain a precipitate, washing the precipitate for 2 times by deionized water and absolute ethyl alcohol respectively, and drying for 10 hours at 40 ℃ to obtain Fe3O4-TiO2 nanoparticles;
s3, dissolving 2.2g of chitosan in 101mL of acetic acid solution with the mass fraction of 2%, stirring for reaction for 12h, adding 0.41g of Fe3O4-TiO2 nanoparticles, 20.3mLspan-80 and 121mLOP-10 emulsifier, uniformly mixing, adding 2.2mL of glutaraldehyde solution with the mass fraction of 25%, heating to 43 ℃ for water bath reaction for 2h, adjusting the pH to 10 by using 1mol/L of sodium hydroxide solution, centrifuging to obtain precipitates, washing the precipitates for 3 times by using deionized water and absolute ethyl alcohol respectively, and drying at 65 ℃ to constant weight to obtain Fe3O4-TiO 2-chitosan powder;
step S4, adding 14.4g of Fe3O4-TiO 2-chitosan powder into 703mL of acetic acid solution with the volume fraction of 1%, swelling for 42h at room temperature, and stirring at the rotating speed of 310rpm until the solution is dissolved to obtain Fe3O4-TiO 2-chitosan solution;
step S5, adding 6.4g of gelatin powder and 103mL of deionized water into a three-neck flask, heating to 60 ℃, and magnetically stirring until the gelatin powder is dissolved to obtain a gelatin solution;
step S6, uniformly mixing the Fe3O4-TiO 2-chitosan solution and the gelatin solution, adding glycerol, uniformly stirring, paving a film, drying in a 50 ℃ oven for 44 hours, and removing the film to obtain a modified composite film, wherein the mass ratio of the Fe3O4-TiO 2-chitosan solution to the gelatin solution is 1:0.45 percent, and the dosage of the glycerol is 15 percent of the total mass of the Fe3O4-TiO 2-chitosan solution and the gelatin solution.
Example 3
The modified composite membrane is prepared by the following steps:
step S1, dispersing 62.5mgFe 3O4 in 45mL of ethanol solution with the mass fraction of 95%, adding 0.4mL of ammonia water with the mass fraction of 28%, performing ultrasonic dispersion for 25min, placing the mixture in a water bath kettle at 45 ℃, stirring and reacting for 2h to obtain mixed solution a, and dissolving 2mL of butyl titanate in 25mL of absolute ethanol to obtain mixed solution b; dropping the mixed solution b into the mixed solution a, carrying out mixing reaction for 26h, then carrying out magnetic separation to obtain solid particles, and washing the solid particles for 3 times by using deionized water and absolute ethyl alcohol respectively to obtain Fe3O4-TiO2 microspheres;
s2, adding 61.5mgFe 3O4-TiO2 microspheres into a reaction kettle, adding 42mL of 1mol/L sodium hydroxide solution, heating to 140 ℃, reacting for 3 hours, centrifuging to obtain a precipitate, washing the precipitate for 3 times by deionized water and absolute ethyl alcohol respectively, and drying at 40 ℃ for 12 hours to obtain Fe3O4-TiO2 nanoparticles;
s3, dissolving 2.3g of chitosan in 103mL of acetic acid solution with the mass fraction of 2%, stirring for reaction for 12 hours, adding 0.42g of Fe3O4-TiO2 nanoparticles, 20.5mL of span-80 and 122mL of emulsifier, uniformly mixing, adding 2.4mL of glutaraldehyde solution with the mass fraction of 25%, heating to 45 ℃ for water bath reaction for 3 hours, adjusting the pH to 10 by using 1mol/L of sodium hydroxide solution, centrifuging to obtain precipitates, washing the precipitates for 3 times by using deionized water and absolute ethyl alcohol respectively, and drying at 70 ℃ to constant weight to obtain Fe3O4-TiO 2-chitosan powder;
s4, adding 14.6g of Fe3O4-TiO 2-chitosan powder into 705mL of acetic acid solution with the volume fraction of 1%, swelling for 48h at room temperature, and stirring at the rotating speed of 320rpm until the chitosan is dissolved to obtain Fe3O4-TiO 2-chitosan solution;
step S5, adding 6.5g of gelatin powder and 105mL of deionized water into a three-neck flask, heating to 60 ℃, and magnetically stirring until the gelatin powder is dissolved to obtain a gelatin solution;
step S6, uniformly mixing the Fe3O4-TiO 2-chitosan solution and the gelatin solution, adding glycerol, uniformly stirring, paving a film, drying in a 50 ℃ oven for 48 hours, and removing the film to obtain a modified composite film, wherein the mass ratio of the Fe3O4-TiO 2-chitosan solution to the gelatin solution is 1:0.45 percent, and the dosage of the glycerol is 15 percent of the total mass of the Fe3O4-TiO 2-chitosan solution and the gelatin solution.
Example 4
A method for culturing eggs of whitmania pigra, which comprises the following steps:
step A1, preparation of a breeding place: building a greenhouse, building a cement breeding pool in the greenhouse, wherein the width of the breeding pool is 0.6m, the height of the breeding pool is 1m, the distance between adjacent breeding pools is 1.3m, carrying out strong chlorine disinfection on the breeding pools, airing for 7 days, paving the modified composite membrane in the breeding pool, adding loess as a production bed, the depth of the loess is 0.6m, and adding fermented farmyard manure, wherein the loess adopts quicklime to remove pests and harmful bacteria in soil, so that damage to a leech breeding place and damage to egg cocoons are avoided, and the using amount is 60 kg/mu;
step A2, selecting leeches: the leech seed is formed by selecting leech which is bright in color, strong, thick, lively and movable and can be quickly shrunk into a ball after being touched by hand;
step A3, disinfecting leeches: disinfecting leeches for 20min, then lightly placing on a production bed of a breeding pond, and covering with straws;
step A4, mating and fertilization: placing leeches, enabling the head ends of the leeches to be opposite in direction, enabling the ventral surfaces of the leeches to lean together, enabling respective male organs to just face the female genital orifice of the other party, then inserting the thin linear penis extending out of the male into the female genital orifice of the other party, and enabling the mating time to last for 2 hours, so that the silence of the surrounding environment is guaranteed during mating;
step A5, hatching egg cocoons: after 30 days, the egg cocoons of the leech seeds are taken out from the obstetric table, 2 egg cocoons of each leech seed are transferred into an incubator, a layer of incubating soil with the thickness of 1.5cm is paved at the bottom of the incubator, the tip egg cocoons are placed on the incubating soil upwards, a layer of incubating soil with the thickness of 1.5cm is covered on the incubating soil, the young leech is incubated for 20 days, the relative humidity of air in the incubator is 75%, and the humidity of the incubating soil is 40%.
Example 5
A method for culturing eggs of whitmania pigra, which comprises the following steps:
step A1, preparation of a breeding place: building a greenhouse, building a cement breeding pool in the greenhouse, wherein the width of the breeding pool is 0.7m, the height of the breeding pool is 1.1m, the distance between adjacent breeding pools is 1.4m, carrying out strong chlorine disinfection on the breeding pools, airing for 8 days, paving the modified composite membrane in the breeding pool, then adding loess as a production bed, the depth of the loess is 0.6m, and then adding fermented farmyard manure, wherein the loess adopts quicklime to remove pests and harmful bacteria in the soil, so that damage to a leech breeding place and damage to egg cocoons are avoided, and the using amount is 70 kg/mu;
step A2, leech seed selection: selecting adult leeches which are bright in color, strong, thick, lively and movable and can be quickly shrunk into a ball after being touched by hands as leeches seeds;
step A3, disinfecting leeches: disinfecting leeches for 20min, then lightly placing on a production bed of a breeding pond, and covering with straws;
step A4, mating and fertilization: placing leeches, enabling the head ends of the leeches to be opposite in direction, enabling the ventral surfaces of the leeches to lean together, enabling respective male organs to just face the female genital orifice of the other party, then inserting the thin linear penis extending out of the male into the female genital orifice of the other party, and enabling the mating time to last for 2 hours, so that the silence of the surrounding environment is guaranteed during mating;
step A5, hatching egg cocoons: after 30 days, the egg cocoons of the leech seeds are taken out from the obstetric table, 2 egg cocoons of each leech seed are transferred into an incubator, a layer of incubating soil with the thickness of 1.5cm is paved at the bottom of the incubator, the tip egg cocoons are placed on the incubating soil upwards, a layer of incubating soil with the thickness of 1.5cm is covered on the incubating soil, the young leech is incubated for 20 days, the relative humidity of air in the incubator is 75%, and the humidity of the incubating soil is 40%.
Example 6
A method for breeding eggs of whitmania pigra, which comprises the following steps:
step A1, preparation of a breeding place: building a greenhouse, building a cement breeding pool in the greenhouse, wherein the width of each breeding pool is 0.8m, the height of each breeding pool is 1.2m, the distance between every two adjacent breeding pools is 1.5m, sterilizing the breeding pools by strong chlorine, airing for 10 days, paving the modified composite membrane in each breeding pool, adding loess as a production bed, the depth of the loess is 0.7m, and adding fermented farmyard manure, wherein the loess adopts quick lime to remove pests and harmful bacteria in soil, so that the damage to a leech breeding place and egg cocoons is avoided, and the using amount is 80 kg/mu;
step A2, selecting leeches: selecting adult leeches which are bright in color, strong, thick, lively and movable and can be quickly shrunk into a ball after being touched by hands as leeches seeds;
step A3, disinfecting leeches: disinfecting leeches for 20min, then lightly placing on a production bed of a breeding pond, and covering with straws;
step A4, mating and fertilization: placing leeches, enabling the head ends of the leeches to be opposite in direction, enabling the ventral surfaces of the leeches to lean together, enabling respective male organs to just face the female genital orifice of the other party, then inserting the thin linear penis extending out of the male into the female genital orifice of the other party, enabling the mating time to last for 3h, and ensuring the quiet surrounding environment during mating;
step A5, hatching egg cocoons: after 30 days, the egg cocoons of the leech seeds are taken out from the obstetric table, 3 egg cocoons of each leech seed are transferred into an incubator, a layer of incubating soil with the thickness of 1.5cm is paved at the bottom of the incubator, the tip egg cocoons are placed on the incubating soil upwards, a layer of incubating soil with the thickness of 1.5cm is covered on the incubating soil, the young leech is incubated for 20 days, the young leech is placed into a fine culture pond, wherein the temperature in the incubator is 27 ℃, the relative humidity of air is 75 percent, and the humidity of the incubating soil is 40 percent.
Comparative example 1
The method for breeding eggs in comparative example 1 is as described in example 2, except that the modified composite membrane is not laid inside the breeding pond.
The following tests were carried out for examples 4-6 and comparative example 1: the number of deaths of young leeches cultivated in examples 4 to 6 and comparative example 1 was counted, and the survival rate was calculated as = (number of released-number of deaths)/number of released × 100%, and the test data are shown in table 1:
TABLE 1
Example 4 example 5 example 6 comparative example 1 survival rate 91% 92% 92% 59%
As can be seen from Table 1, the survival rate of the young leeches incubated in examples 4 to 6 was as high as 92% compared with that of comparative example 1.
The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise embodiments disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best understand the invention for and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.
Claims (3)
1. The method for breeding eggs of whitmania pigra, which is characterized by comprising the following steps:
step A1, preparation of a breeding place: building a greenhouse, building a cement breeding pool in the greenhouse, sterilizing the breeding pool by strong chlorine, airing for 7-10 days, paving the modified composite membrane in the breeding pool, adding loess as a obstetric table, and adding fermented farmyard manure;
step A2, selecting leeches: selecting adult leeches as seed leeches;
step A3, disinfecting leeches: disinfecting leeches for 20min, then placing the leeches on a production bed of a breeding pond, and covering straws with the breeding pond;
step A4, mating and fertilization: placing the leeches, enabling the head ends of the leeches to be opposite in direction, enabling the ventral surfaces of the leeches to lean together, mating, and enabling mating time to last for 2-3 hours;
step A5, hatching egg cocoons: after 30 days, the egg cocoons of the leech seeds are taken out from the obstetric table and transferred into an incubator, a layer of 1.5 cm-thick hatching soil is paved at the bottom of the incubator, the sharp egg cocoons are placed upwards on the hatching soil, a layer of 1.5 cm-thick hatching soil is covered on the top of the incubator, the young leeches begin to grow after 20 days of incubation, and the young leeches are placed into a fine culture pond;
the modified composite membrane is prepared by the following steps:
step S1, dispersing Fe3O4 in an ethanol solution, adding ammonia water, performing ultrasonic dispersion, then placing the mixture in a 45 ℃ water bath kettle, stirring and reacting for 1-2 hours to obtain a mixed solution a, dissolving butyl titanate in absolute ethyl alcohol to obtain a mixed solution b, dripping the mixed solution b into the mixed solution a, performing mixed reaction for 24-26 hours, separating to obtain solid particles, and washing the solid particles to obtain Fe3O4-TiO2 microspheres;
s2, uniformly stirring the Fe3O4-TiO2 microspheres and a sodium hydroxide solution, heating to 140 ℃, reacting for 2-3h, centrifuging to obtain a precipitate, and washing and drying the precipitate to obtain Fe3O4-TiO2 nano particles;
step S3, dissolving chitosan in an acetic acid solution, stirring and reacting for 12 hours, adding Fe3O4-TiO2 nanoparticles, span-80 and OP-10 emulsifier, uniformly mixing, adding a glutaraldehyde solution, heating to 40-45 ℃, reacting in a water bath for 2-3 hours, adjusting the pH to 9-10 by using a sodium hydroxide solution, centrifuging to obtain a precipitate, washing and drying the precipitate to obtain Fe3O4-TiO 2-chitosan powder;
step S4, adding Fe3O4-TiO 2-chitosan powder into an acetic acid solution, swelling for 36-48h at room temperature, and stirring until dissolving to obtain a Fe3O4-TiO 2-chitosan solution;
step S5, adding gelatin powder and deionized water into a three-neck flask, heating to 60 ℃, and stirring until the gelatin powder and the deionized water are dissolved to obtain a gelatin solution;
and S6, uniformly mixing the Fe3O4-TiO 2-chitosan solution and the gelatin solution, adding glycerol, uniformly stirring, paving the film, and drying in a 50 ℃ oven for 40-48 hours to obtain the modified composite film.
2. The method for breeding eggs of whitmania pigra according to claim 1, which is characterized in that: the width of the breeding pools in the step A1 is 0.6-0.8m, the height is 1-1.2m, the distance between every two adjacent breeding pools is 1.3-1.5m, the depth of the loess is 0.6-0.7m, and the loess is disinfected by quicklime.
3. The method for breeding eggs of whitmania pigra according to claim 1, which is characterized in that: the temperature in the incubator in the step A5 is 20-27 ℃, the relative humidity of air is 75%, and the humidity of the hatching soil is 40%.
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