CN101427658B - Artificial insemination incubation method for sepiella maindroni and special agent used thereof - Google Patents
Artificial insemination incubation method for sepiella maindroni and special agent used thereof Download PDFInfo
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- CN101427658B CN101427658B CN2008101218978A CN200810121897A CN101427658B CN 101427658 B CN101427658 B CN 101427658B CN 2008101218978 A CN2008101218978 A CN 2008101218978A CN 200810121897 A CN200810121897 A CN 200810121897A CN 101427658 B CN101427658 B CN 101427658B
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- nutrient solution
- sepiella maindroni
- seawater
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- 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
Abstract
The invention discloses a cuttle-fish artificial insemination and incubation method and special agent thereof, wherein the eggs in oviduct of the female cuttle-fish are transferred into a container with nutrient solution to perform external insemination and isolated culture and an agarose layer is set between the container bottom and eggs; the nutrient solution contains the fresh or freeze-dry oviduct gland secretion of the female cuttle-fish. The special agent contains culture container bottom liner and nutrient solution and the culture container bottom liner is 0.2% of agarose and the nutrient solution contains 1-10 portions by weight of blood serum and 1000 portions by weight of sterile sea water. Because the molecular structure of agarose is loose, the nutrient material can freely flow and penetrate, the eggs on the agarose liner has little distortion without nutrition deficiency of the contact surface, The nutrition is provided by the oviduct gland secretion and the blood serum of low concentration, the eggs do not distort, thus the whole development process of the cuttle-fish embryo cultured in vitro is greatly accomplished.
Description
Technical field
The present invention relates to a kind of aquatic livestock artificial insemination incubation method and special-purpose reagent, particularly a kind of Sepiella maindroni artificial insemination incubation method and the special agent thereof of hatching thereof.
Background technology
Sepiella maindroni once was one of the four big fishery in China fishing ground, and was stronger to the adaptability of temperature, in China coastal distribution very wide, be the very high traditional fishery kind of a kind of economic worth.Because overfishing and ecological degeneration since the seventies in 20th century, decay successively occurred.Last century, the eighties had the Primary Study to Sepiella maindroni resource and propagation such as report Li Xing a word used in person's names, and abroad had the biology of merchant cuttlefish and the report of breed aspect.Carry out the cuttlefish artificial propagation and grow seedlings, at first must carry out the research of the fundamental biological knowledge of cuttlefish.And the research of the fundamental biological knowledge of cuttlefish is needed the support of the research of reliable embryonic development.Only after abundant understanding, could further carry out artificial breeding and the research of growing seedlings to embryo development procedure.And to the research of embryonic development, optimum way is carried out culture in vitro and observation to its embryo exactly.
The basic research of relevant siphonopods early development stage, report all seldom both at home and abroad.Artificial insemination is hatched to Sepiella maindroni research especially beyond example.Such research is intended to be siphonopods fundamental biological knowledge research stockpile, for the seed of Sepiella maindroni produces and the raising of survival rate provides theoretical foundation.On this basis, can further intervene its embryo, make and realize that further production purpose becomes possibility.
Summary of the invention
The purpose of this invention is to provide a kind of Sepiella maindroni artificial insemination incubation method and special agent thereof, realize technical problem with this, and this method of operating is simple the stripped embryo culture of Sepiella maindroni, with low cost.
The present invention is achieved in that a kind of Sepiella maindroni artificial insemination incubation method, its will be in the female gonosome of Sepiella maindroni the ovum the oviduct move in the container that nutrient solution is housed and carry out in inseminatio externalis and cultured in vitro, and be provided with agarose layer between container bottom and the ovum; Nutrient solution contains oviduct glandular secretion liquid fresh or the female Sepiella maindroni body that freeze-drying is handled.
The spermatophore of male Sepiella maindroni is adopted in the sperm source of inseminatio externalis; Spermatophore is placed on the agarose layer, and extruding is to discharge sperm; Nutrient solution also contains serum.
This method can be operated as follows:
1. choose Sepiella maindroni and gather ovum and spermatophore, artificial seawater cleans stand-by; In culture vessel, put into the artificial seawater that is mixed with oviduct glandular secretion liquid;
2. the sheet agarose layer is placed bottom of culture vessel; Step ovum 1. is placed on the agarose layer, puts into spermatophore and extruding spermatophore, carry out inseminatio externalis; Cultivating temperature is 26 ± 1 ℃, changes nutrient solution and removes discarded spermatophore every 0.5 hour to 3 hours;
3. after being fertilized 1 day to 2 days, in nutrient solution, add the serum of horse or ox; Take out fertilized egg every 15 hours to 30 hours and change to new culture vessel; Nurturing period is after fertilization 12 days to 14 days.
The 1. middle artificial seawater of step was exposed to the sun three days at least, and artificial seawater is the good seawater of inflation through micro porous filtration; The salinity of artificial seawater is 27 ‰ to 28 ‰.
Also contain EDTA in the artificial seawater, and in the artificial seawater concentration of EDTA less than 3 gram/rice
3
Special agent is hatched in a kind of Sepiella maindroni artificial insemination, and it contains culture vessel end liner and nutrient solution; The culture vessel end liner is 0.2% agarose; Nutrient solution contains 1 part to 10 parts serum and 1000 parts aseptic seawater by weight.
Aseptic seawater is the good seawater of inflation through micro porous filtration.
Aseptic seawater also contains EDTA, and the concentration of EDTA is less than 3 gram/rice
3
Compared with prior art, the invention has the advantages that:
The present invention has used the cultural method of agarose substrate, in conjunction with the artificial seawater that serum, oviduct glandular secretion liquid mix, ground-breakingly is applied to Sepiella maindroni artificial insemination and hatches.Because training method of traditional linerless end causes the distortion of ovum because the container bottom quality is hard, and egg membrane and container bottom contact-making surface can not absorb nutriment, can cause embryonic development deformity even growth to stop, and culture success ratio is extremely low.The technical program is because the agarose molecular structure is loose, and nutriment can permeate in free-flow, and ovum is out of shape less on the agarose substrate, and can not cause the nutritional deficiency of contact-making surface.Under the situation that oviduct glandular secretion liquid or serum use separately, because the disappearance of crucial nutritive element still the phenomenon that embryonic development stops can to occur.Only being equipped with low concentration serum at oviduct glandular secretion liquid provides under the condition of nutrition, and guarantees that ovum is indeformable, just can make the embryo of Sepiella maindroni culture in vitro finish whole growth course smoothly.
Description of drawings
Fig. 1 is the microphoto of each developmental stage of embryo of embodiment of the invention cultivation.
Embodiment
Below in conjunction with attached embodiment the present invention is described in further detail.
Embodiment: special agent is hatched in a kind of Sepiella maindroni artificial insemination, and it contains culture vessel end liner and nutrient solution; The culture vessel end liner is 0.2% agarose; Nutrient solution contains 1 part serum or 1 part of oviduct glandular secretion liquid or its combination by weight; Nutrient solution also contains 500 parts aseptic seawater by weight.At the cultivation stage of fertilization before 2 days, use the oviduct glandular secretion liquid of 1 part Sepiella maindroni separately, then adopt 1 part of serum to add 1 part of oviduct glandular secretion liquid after 2 days as the culture fluid that nutrition is provided.Test shows so just can make the embryo normally finish whole growth course.
Aseptic seawater is the good seawater of inflation through micro porous filtration, promptly by the manual inflation, makes its oxygen content be not less than the oxygen content of common nature seawater at least.Also contain EDTA in the aseptic seawater, and the concentration of EDTA is less than 3 gram/rice
3
A kind of Sepiella maindroni artificial insemination incubation method, will be in the female gonosome of Sepiella maindroni the ovum the oviduct move in the plastic culture dish that nutrient solution is housed and carry out in inseminatio externalis and cultured in vitro, and be provided with agarose layer between plastic culture dish bottom and the ovum; Nutrient solution contains oviduct glandular secretion liquid fresh or the female Sepiella maindroni body that freeze-drying is handled.Agarose layer is preparation in advance in glass culture dish, moves in the plastic culture dish during use, is tiled in the bottom.
The spermatophore of male Sepiella maindroni is adopted in the sperm source of inseminatio externalis; Spermatophore is placed on the agarose layer, and extruding is to discharge sperm; Nutrient solution also contains serum.
The concrete steps of this method are as follows:
1. preparation: choose Sepiella maindroni and gather ovum and spermatophore, artificial seawater cleans stand-by; In cultivating plastic culture dish, put into the artificial seawater that is mixed with oviduct glandular secretion liquid.Test shows the early development that the nutrient solution of this moment can embryo support, yet the phenomenon that stops with embryonic death can appear growing in the later stage.Cuttlefish natural disposition ferociousness is preferably anaesthetized earlier when operation, gathers the ovum of ripe bright in color from its oviduct or ovary.
2. artificial insemination: the sheet agarose layer is placed cultivation plastic culture dish bottom; Step ovum 1. is placed on the agarose layer, puts into spermatophore and extruding spermatophore, carry out inseminatio externalis; After fertilization is abandoned spermatophore when changing nutrient solution, change nutrient solution every half an hour; Cultivating temperature is 26 ± 1 ℃.
3. after being fertilized 2 days, increase the serum of horse or ox on the basis of former nutrient solution, increasing the needed nutrition of embryonic development, otherwise the embryo mortality can occur when growing in the later stage; Take out fertilized egg every 24 hours and change to new cultivation plastic culture dish, and remove dead ovum; Nurturing period is after fertilization 12 days to 14 days, and Sepiella maindroni is at broken ovum after growing in 12 days to 14 days generally speaking, and whole cultivating process has also just been finished thereupon.
Than higher, sterile working can improve embryo's survival rate to a certain extent to hygienic requirements in all operations.
Step 1. in artificial seawater be exposed to the sun at least three days, and artificial seawater is that the good salinity of inflation through micro porous filtration is about 27.5 ‰ seawater.
Also contain EDTA in the artificial seawater, and in the artificial seawater concentration of EDTA less than 3 gram/rice
3Here EDTA is an ion chelating agent, can the chelating heavy metal ion, prevent the murder by poisoning of heavy metal ion too much in the seawater to the embryo.
The cultural method that uses the agarose substrate of the present invention in conjunction with the artificial seawater that serum, oviduct glandular secretion liquid mix, ground-breakingly is applied to Sepiella maindroni artificial insemination and hatches.Because training method of traditional linerless end causes the distortion of ovum because the container bottom quality is hard, and egg membrane and container bottom contact-making surface can not absorb nutriment, can cause embryonic development deformity even growth to stop, and culture success ratio is extremely low.The technical program is because the agarose molecular structure is loose, and nutriment can permeate in free-flow, and ovum is out of shape less on the agarose substrate, and can not cause the nutritional deficiency of contact-making surface.Under the situation that oviduct glandular secretion liquid or serum use separately, because the disappearance of crucial nutritive element still the phenomenon that embryonic development stops can to occur.Only being equipped with low concentration serum at oviduct glandular secretion liquid provides under the condition of nutrition, and guarantees that ovum is indeformable, just can make the embryo of Sepiella maindroni culture in vitro finish whole growth course smoothly.Can make things convenient for the manual intervention embryonic development in culture in vitro, thereby realize further industrialization purpose.
Although described the present invention in conjunction with preferred embodiment; right its is not in order to limit the present invention; any those skilled in the art; under the situation that does not break away from the spirit and scope of the present invention; can implement the displacement and the modification of various changes, coordinate to the theme of listing here, so protection scope of the present invention is as the criterion when looking the claim restricted portion that is proposed.
Claims (8)
1. Sepiella maindroni artificial insemination incubation method, it is characterized in that: will be in the female gonosome of Sepiella maindroni the ovum the oviduct move in the container that nutrient solution is housed and carry out in inseminatio externalis and cultured in vitro, and be provided with agarose layer between described container bottom and the ovum; Described nutrient solution contains oviduct glandular secretion liquid fresh or the female Sepiella maindroni body that freeze-drying is handled; After fertilization adds horse or cow's serum and supplements the nutrients.
2. a kind of Sepiella maindroni artificial insemination incubation method according to claim 1 is characterized in that: the spermatophore of male Sepiella maindroni is adopted in the sperm source of inseminatio externalis; Described spermatophore is placed on the described agarose layer, and extruding is to discharge sperm; Described nutrient solution also contains serum.
3. a kind of Sepiella maindroni artificial insemination incubation method according to claim 2 is characterized in that having following steps: 1. choose Sepiella maindroni and gather ovum and spermatophore, artificial seawater cleans stand-by; In culture vessel, put into the artificial seawater that is mixed with oviduct glandular secretion liquid;
2. the sheet agarose layer is placed described bottom of culture vessel; Step ovum 1. is placed on the described agarose layer, puts into spermatophore and extruding spermatophore, carry out inseminatio externalis; Cultivating temperature is 26 ± 1 ℃, changes nutrient solution and removes discarded spermatophore every 0.5 hour to 3 hours;
3. after being fertilized 1 day to 2 days, in nutrient solution, add the serum of horse or ox; Take out fertilized egg every 15 hours to 30 hours and change to new culture vessel; Nurturing period is after fertilization 12 days to 14 days.
4. a kind of Sepiella maindroni artificial insemination incubation method according to claim 3 is characterized in that: the artificial seawater of step described in 1. was exposed to the sun three days at least, and described artificial seawater is the good seawater of inflation through micro porous filtration; The salinity of described artificial seawater is 27 ‰ to 28 ‰.
5. a kind of Sepiella maindroni artificial insemination incubation method according to claim 4 is characterized in that: also contain EDTA in the described artificial seawater, and in the described artificial seawater concentration of EDTA less than 3 gram/rice
3
6. special agent is hatched in a Sepiella maindroni artificial insemination, it is characterized in that: contain culture vessel end liner and nutrient solution; Described culture vessel end liner is 0.2% agarose; Described nutrient solution contains 1 part of serum by weight and adds 1 part of oviduct glandular secretion liquid; Described nutrient solution also contains 1000 parts aseptic seawater by weight.
7. special agent is hatched in a kind of Sepiella maindroni artificial insemination according to claim 6, it is characterized in that: described aseptic seawater is the good seawater of inflation through micro porous filtration.
8. special agent is hatched in a kind of Sepiella maindroni artificial insemination according to claim 6, it is characterized in that: described aseptic seawater also contains EDTA, and the concentration of EDTA is less than 3 gram/rice
3
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| CN2008101218978A CN101427658B (en) | 2008-10-21 | 2008-10-21 | Artificial insemination incubation method for sepiella maindroni and special agent used thereof |
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| CN2008101218978A CN101427658B (en) | 2008-10-21 | 2008-10-21 | Artificial insemination incubation method for sepiella maindroni and special agent used thereof |
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| CN101427658B true CN101427658B (en) | 2010-09-08 |
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| CN103314900B (en) * | 2013-06-17 | 2016-04-20 | 苏州市阳澄湖现代农业发展有限公司 | A kind of Sepiella maindroni pond high-efficiency breeding method |
| CN106719178B (en) * | 2016-12-05 | 2020-06-09 | 浙江海洋大学 | Artificial ripening method for cephalopods |
| CN106900600B (en) * | 2016-12-30 | 2020-11-13 | 浙江海洋大学 | Enhanced cultivation method for improving fertility of sepiella maindroni |
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Granted publication date: 20100908 Termination date: 20121021 |