CN104798708B - Maturity promoting and artificial fertilization method for high-quality ova of starfishes - Google Patents
Maturity promoting and artificial fertilization method for high-quality ova of starfishes Download PDFInfo
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- CN104798708B CN104798708B CN201510178843.5A CN201510178843A CN104798708B CN 104798708 B CN104798708 B CN 104798708B CN 201510178843 A CN201510178843 A CN 201510178843A CN 104798708 B CN104798708 B CN 104798708B
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- ovum
- asterias amurensis
- amurensis lutken
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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 provides a maturity promoting and artificial fertilization method for high-quality ova of starfishes. The method comprises the following steps of (1) starfish parent culture; (2) starfish male and female gonad tissue collection; (3) adoption of 1-O6-methylguanine with the concentration being 2mum for starfish ovum maturity promotion; (4) artificial fertilization of the starfishes. The invention builds the obtaining and artificial fertilization method for the high-quality ova of the starfishes for the first time, and through experiments, proper maturity promoting agents and maturity promoting time of the starfish ova are obtained through screening. The method has the characteristics that the operation is convenient, the efficiency is high, and the practicability is realized. Parents cannot be injured, and the ovum obtaining is not limited by seasons. Important application values and popularization prospects are realized in the aspects of echinodermata animal reproduction biology and experiment embryology study.
Description
Technical field
The invention belongs to field of marine biotechnology, and in particular to a kind of accelerating of Asterias amurensis Lutken high-quality ovum and artificial fertilization side
Method.
Background technology
Asterias amurensis Lutken is the relatively common invertebratess of a class, is the representative clock of echinoderm, with becoming model animal
Advantage：Individual relative is less, vitality is strong, wide adaptability, easily raise under laboratory control condition；Breeding cycle phase
To short, can control in laboratory conditions；Requirement can be laid eggs in batches or continuously be laid eggs, and have certain egg laying amount；Pollution-free
Without hermaphroditism under natural conditions.Early in 1882, Ai Liyeailihemeiqi Elie Metchnikoffs just entered to Asterias amurensis Lutken larva
Row research, is found that phagocyte for the first time and obtains Nobel prize's soul in 1908.Therefore, Asterias amurensis Lutken is to carry out biology
The good experimental material of research is learned, moreover, Asterias amurensis Lutken has higher medicinal health and a nutritive value, its living resources is opened
Send out to utilize and be also subject to people's attention.
Sea urchin embryos are easy to observe and operate, and are to carry out micrurgy, carry out the good material of reproductive development biology, therefore
And the acquisition of its high-quality ovum is particularly important.Acquisition and Artificial insemination procedures with regard to Asterias amurensis Lutken high-quality ovum is had no both at home and abroad
Report.
The content of the invention
The invention provides a kind of accelerating of Asterias amurensis Lutken high-quality ovum and Artificial insemination procedures.The present invention establishes a kind of quick
The Artificial insemination procedures of Asterias amurensis Lutken ovum and the simplicity of accelerating the ripening.
For achieving the above object, the present invention is achieved using following technical proposals：
A kind of accelerating of Asterias amurensis Lutken high-quality ovum and Artificial insemination procedures, it comprises the following steps：
（1）The cultivation of Asterias amurensis Lutken parent
Asterias amurensis Lutken parent is placed in the aquarium of 15～16 DEG C of constant temperature and is cultivated, during culture, feed microzooplankton, 2～
Feed once within 3 days, change water 100% within 4～6 days；
（2）The collection of Asterias amurensis Lutken gonadal tissue
The seminal fluid in the ovum liquid and male gonad in Asterias amurensis Lutken arm female gonads is gathered respectively；
（3）The artificial ripening of Asterias amurensis Lutken ovum
The 1- methyl guanines of final concentration of 2 M are added in the ovum liquid for collecting, is placed in 16 DEG C of incubators, use
Shaking table shakes maturation of ovum after 1～2h；It is standby using fresh seawater washing ovum；
（4）Artificial fertilization
The seminal fluid sea water for collecting is diluted into 50～100 times and obtains spermatozoa diluent, draw the spermatozoa diluent
It is added to 2 × 103～5 × 103In individual ovum, 30～50mL sea water is added, be shaken gently for 5～10 minutes, seen under anatomical lens
Fertilization effect is examined, egg membrane is lifted, show that fertilization is completed.
Further improvement to above-mentioned technical proposal：The step（1）In initial water temperature be 12 DEG C, using electrical bar plus
Temperature, heats up 0.5～1 DEG C daily, maintains this temperature during to 16 DEG C.
Further improvement to above-mentioned technical proposal：The step（1）Middle-size and small-size zooplankton is artemia and/or small-sized oar
Sufficient class.
Further improvement to above-mentioned technical proposal：The scale of feeding of the microzooplankton is every 0.5～1g of Asterias amurensis Lutken.
Further improvement to above-mentioned technical proposal：The step（2）The taper draw point of 2～3mm of middle employing diameter is in sea
The aperture of an a diameter of 2～3mm is gently opened on one arm of star, well-developed a small amount of gonad is taken out with elbow tweezers, will
The female gonads of taking-up are put in a little sea water, are placed on ice, are obtained Asterias amurensis Lutken ovum after gauze is filtered；The male that will be taken out
Gonadal tissue shreds centrifugation, and it is seminal fluid to take supernatant fluid, is placed on ice.
Further improvement to above-mentioned technical proposal：The gonad for female and shows that gonad development is good in Chinese red；
Gonad is creamy white as male and shows that gonad development is good.
Further improvement to above-mentioned technical proposal：The step（3）In middle ovum liquid, the concentration of ovum is 1 × 103~2×
103Individual/mL.
Further improvement to above-mentioned technical proposal：The step（4）The concentration of middle spermatozoa diluent is 1 × 105~2×
106Individual/mL.
Compared with prior art, advantages of the present invention and good effect are：The present invention is by experiment screening to suitable for sea
The most suitable maturing agent of star ovum and most suitable accelerating time, the mature egg of high-quality can be obtained rapidly using the maturing agent, and not
Any impact can be produced on operations such as follow-up fertilizations, so as to establish a kind of quick acquisition Asterias amurensis Lutken high-quality ovum and artificial fertilization
Method；And, the present invention will not cause very major injury and dead to Asterias amurensis Lutken parent, and parent can be used for multiple times, the acquisition of ovum
Be not subject to seasonal restrictions, can accomplish on demand for ovum, good experiment material be provided for carrying out biology of reproduction and genetics research
Material.
Description of the drawings
Fig. 1 is the Asterias amurensis Lutken ovum form before and after embodiment of the present invention accelerating, and wherein Figure 1A is the sea star egg subgraph before accelerating,
Figure 1B is the sea star egg subgraph after accelerating.
Specific embodiment
Technical scheme is described in further detail below in conjunction with the drawings and specific embodiments.
The accelerating of Asterias amurensis Lutken high-quality ovum of the present invention and Artificial insemination procedures comprise the following steps：
1st, the cultivation of Asterias amurensis Lutken parent
By randomly select 20 ripe Asterias amurensis Lutken parents（The quantity of female parent and male parent is determined according to gonad）Put
Cultivate in 100L aquariums, initial water temperature is 12 DEG C, is heated using electrical bar, is heated up 0.5～1 DEG C daily, is tieed up to when 16 DEG C
Hold this temperature.Microzooplankton such as artemia and Small Copepods is fed during culture, feeds 10～20g every time（Every Asterias amurensis Lutken
0.5～1g）, feeding within 2-3 days 1 time, changed water 100% every 6 days, parent culture is more than one week.
2nd, the collection of Asterias amurensis Lutken gonadal tissue
One aperture is gently opened on one arm of Asterias amurensis Lutken using the taper draw point of diameter 3mm, with elbow tweezers by a small amount of property
Gland takes out（Because Asterias amurensis Lutken has 5 Asterias amurensis Lutken arms, every Asterias amurensis Lutken arm all has gonadal tissue, while Asterias amurensis Lutken also has very strong regeneration
Ability, only taking a small amount of gonad from 1～2 Asterias amurensis Lutken arm can't damage to Asterias amurensis Lutken, Asterias amurensis Lutken by means of its rapid regeneration ability,
Original state is reverted to rapidly can, so a small amount of gonadal tissue of collection is to its biotic influence very little, will not be individual to Asterias amurensis Lutken
Damage）.Gonad for female and shows that gonad development is good in Chinese red；The a small amount of female gonads for taking out are put in equipped with few
Perhaps in the beaker of sea water, it is placed on ice, obtains the sea star egg liquid containing Asterias amurensis Lutken ovum from female gonads.Gonad be creamy white for
Male and show that gonad development is good, by taking-up after a small amount of male gonad be put in 1.5mL centrifuge tubes, shredded with shears, taken
Supernatant fluid is Asterias amurensis Lutken seminal fluid, is placed on ice.
3rd, the artificial ripening of Asterias amurensis Lutken ovum
Take 5mL sea star egg liquid（In ovum liquid, the concentration of ovum is 1 × 103~2×103Individual/mL）It is placed in the 50mL equipped with sea water
In centrifuge tube, add 1- methyl guanines to make its final concentration of 2 M, be placed in 16 DEG C of incubators, constantly shaken with shaking table, after 1h
Maturation of ovum.Ovum is rinsed with fresh seawater, ovum is placed in 50mL centrifuge tubes after flushing, ovum is washed using fresh seawater
It is standby.Ovum in the present invention can also carry out accelerating using existing immature ovum.
The creative function can there is provided 1- methyl guanines with accelerating Asterias amurensis Lutken ovum of Jing experiments of the present invention, and
The usage amount of 1- methyl guanines also has vital effect, and only suitable consumption can just play effective accelerating Asterias amurensis Lutken
The effect of ovum, consumption it is too high or too it is low all do not have accelerating effect.Fig. 1 is using the Asterias amurensis Lutken before and after 1- methyl guanine acceleratings
Ovum aspect graph, can see that ovum shows slightly irregular before accelerating, has in ovum from the micro- sem observation photo of Figure 1A and Figure 1B
Reproduction is steeped, and the ovum after accelerating is rounded, and reproduction bubble disappears.
4th, artificial fertilization
Take dilution during 10 L Asterias amurensis Lutken seminal fluid are put in 1mL sea water and obtain spermatozoa diluent（Spermatozoa diluent Jing after dilution shows
Micro mirror observation about concentration is 5 × 105Individual/mL）, take 300 L described in spermatozoa diluent be added to 5 × 103In ovum, gently shake
Dynamic, after 5～10 minutes, fertilization process is completed, and it is 95.8% to count rate of fertilization under anatomical lens, after the completion of fertilization, using fresh seawater
Germ cell is rinsed, it is standby.
Above example is only illustrating technical scheme, rather than is limited；Although with reference to aforementioned reality
Apply example to be described in detail the present invention, for the person of ordinary skill of the art, still can be to aforementioned enforcement
Technical scheme described in example is modified, or carries out equivalent to which part technical characteristic；And these are changed or replace
Change, do not make the essence of appropriate technical solution depart from the spirit and scope of claimed technical solution of the invention.
Claims (8)
1. a kind of accelerating of Asterias amurensis Lutken high-quality ovum and Artificial insemination procedures, it is characterised in that it comprises the following steps：
（1）The cultivation of Asterias amurensis Lutken parent
Asterias amurensis Lutken parent is placed in the aquarium of 15～16 DEG C of constant temperature and is cultivated, during culture, feed microzooplankton, 2～3 days
Feed once, change water 100% within 4～6 days；
（2）The collection of Asterias amurensis Lutken gonadal tissue
The seminal fluid in the ovum liquid and male gonad in Asterias amurensis Lutken arm female gonads is gathered respectively；
（3）The artificial ripening of Asterias amurensis Lutken ovum
The 1- methyl guanines of final concentration of 2 M are added in the ovum liquid for collecting, and are placed in 16 DEG C of incubators, are used shaking table
Maturation of ovum after 1～2h of shake；It is standby using fresh seawater washing ovum；
（4）Artificial fertilization
The seminal fluid sea water for collecting is diluted into 50～100 times and obtains spermatozoa diluent, drawn the spermatozoa diluent and add
To 2 × 103～5 × 103In individual ovum, 30～50mL sea water is added, be shaken gently for 5～10 minutes, received Microscopic observation is dissected
Smart effect, egg membrane are lifted, and show that fertilization is completed.
2. the accelerating of Asterias amurensis Lutken high-quality ovum according to claim 1 and Artificial insemination procedures, it is characterised in that：The step
（1）In initial water temperature be 12 DEG C, heated using electrical bar, heat up 0.5～1 DEG C daily, during to 16 DEG C, maintain this temperature.
3. the accelerating of Asterias amurensis Lutken high-quality ovum according to claim 1 and Artificial insemination procedures, it is characterised in that：The step
（1）Middle-size and small-size zooplankton is artemia and/or Small Copepods.
4. the accelerating of the Asterias amurensis Lutken high-quality ovum according to claim 1 or 3 and Artificial insemination procedures, it is characterised in that：It is described
The scale of feeding of microzooplankton is every 0.5～1g of Asterias amurensis Lutken.
5. the accelerating of Asterias amurensis Lutken high-quality ovum according to claim 1 and Artificial insemination procedures, it is characterised in that：The step
（2）The taper draw point of 2～3mm of middle employing diameter gently opens the aperture of an a diameter of 2～3mm on one arm of Asterias amurensis Lutken, with curved
Well-developed a small amount of gonad is taken out by head tweezers, the female gonads of taking-up is put in a little sea water, is placed on ice, by yarn
Asterias amurensis Lutken ovum is obtained after net filtration；The male gonad tissue of taking-up is shredded into centrifugation, it is seminal fluid to take supernatant fluid, is placed on ice.
6. the accelerating of Asterias amurensis Lutken high-quality ovum according to claim 5 and Artificial insemination procedures, it is characterised in that：The gonad
In Chinese red for female and show that gonad development is good；Gonad is creamy white as male and shows that gonad development is good.
7. the accelerating of Asterias amurensis Lutken high-quality ovum according to claim 1 and Artificial insemination procedures, it is characterised in that：The step
（3）In middle ovum liquid, the concentration of ovum is 1 × 103~2×103Individual/mL.
8. the accelerating of Asterias amurensis Lutken high-quality ovum according to claim 1 and Artificial insemination procedures, it is characterised in that：The step
（4）The concentration of middle spermatozoa diluent is 1 × 105~2×106Individual/mL.
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| 受精的机制;adieyou123;《百度文库＞教育专区＞高等教育＞理学http://wenku.baidu.com/view/da44f7c32cc58bd63186bd31.html?from=search》;20120623;1-45 * |
| 海星卵和海胆卵的人工受精及其早期发育的观察方法;武兆发;《生物学通报》;19550630(第6期);第39-41页 * |
| 海星资源的开发现状及发展动态;汪行舟等;《饲料研究》;20080215(第3期);第64-66页 * |
| 虾夷砂海星化学成分的研究;桂丽萍等;《南开大学学报（自然科学版）》;20111231;第44卷(第6期);第99-101、104页 * |
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