CN104798708A - 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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- CN104798708A CN104798708A CN201510178843.5A CN201510178843A CN104798708A CN 104798708 A CN104798708 A CN 104798708A CN 201510178843 A CN201510178843 A CN 201510178843A CN 104798708 A CN104798708 A CN 104798708A
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- starfish
- ovum
- accelerating
- artificial insemination
- insemination procedures
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
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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, be specifically related to a kind of accelerating and Artificial insemination procedures of starfish high-quality ovum.
Background technology
Starfish is the more common invertebrate of a class, is the representative clock of echinoderm, has the advantage becoming model animal: individual relative is less, vitality is strong, wide adaptability, easily raise under the controlled condition of laboratory; Breeding cycle is relatively short, can control in laboratory conditions; Requirement can be laid eggs in batches or lay eggs continuously, has certain egg laying amount; Without hermaphroditism under pollution-free natural conditions.As far back as 1882, Ai Liyeailihemeiqi Elie Metchnikoff was just studied starfish larva, had found phagocyte for the first time and had obtained Nobel prize's soul in 1908.Therefore, starfish is the good experiment material of carrying out biological study, and moreover, starfish has higher medicinal health and nutritive value, and the exploitation of its living resources are also subject to people's attention.
Sea urchin embryos is easy to observe and operation, is carry out micromanipulation, carry out the biological good material of reproductive development, so the acquisition of its high-quality ovum seems particularly important.Report is had no both at home and abroad about the acquisition of starfish high-quality ovum and Artificial insemination procedures.
Summary of the invention
The invention provides a kind of accelerating and Artificial insemination procedures of starfish high-quality ovum.The present invention establishes a kind of rapid accelerating ripening starfish ovum and easy Artificial insemination procedures.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
The accelerating of starfish high-quality ovum and an Artificial insemination procedures, it comprises the following steps:
(1) cultivation of starfish parent
Cultivate in aquarium starfish parent being placed in 15 ~ 16 DEG C of constant temperature, microzooplankton of throwing something and feeding between culture period, throws something and feeds once in 2 ~ 3 days, within 4 ~ 6 days, changes water 100%;
(2) collection of starfish gonadal tissue
Gather the ovum liquid in starfish arm female gonads and the seminal fluid in male gonad respectively;
(3) artificial ripening of starfish ovum
In the described ovum liquid collected, add the 1-methyl guanine that final concentration is 2 μMs, be placed in 16 DEG C of incubators, with maturation of ovum after shaking table shake 1 ~ 2h; Adopt fresh seawater washing ovum for subsequent use;
(4) artificial insemination
The described seminal fluid seawater dilution 50 ~ 100 times collected is obtained spermatozoa diluent, draws described spermatozoa diluent and join 2 × 10
3~ 5 × 10
3in individual ovum, then add 30 ~ 50mL seawater, shake 5 ~ 10 minutes gently, in dissection Microscopic observation fertilization effect, egg membrane lifts, and shows that fertilization completes.
Further improvement to technique scheme: in described step (1), initial water temperature is 12 DEG C, adopts electrical bar to heat, heats up 0.5 ~ 1 DEG C every day, maintain this temperature to when 16 DEG C.
Further improvement to technique scheme: described step (1) middle-size and small-size zooplankton is halogen worm and/or Small Copepods.
Further improvement to technique scheme: the scale of feeding of described microzooplankton is every starfish 0.5 ~ 1g.
Further improvement to technique scheme: adopting the taper draw point of diameter 2 ~ 3mm to open a diameter gently in described step (2) on starfish one arm is the aperture of 2 ~ 3mm, with elbow tweezers, well-developed a small amount of sexual gland is taken out, the female gonads of taking-up is put in a little seawater, be placed on ice, after being filtered by gauze, obtain starfish ovum; Shred centrifugal by the male gonad tissue of taking-up, getting supernatant fluid is seminal fluid, is placed on ice.
Further improvement to technique scheme: described sexual gland is Chinese red to be female and to show that gonad development is good; Sexual gland is creamy white as male and show that gonad development is good.
Further improvement to technique scheme: in described step (3), in ovum liquid, the concentration of ovum is 1 × 10
3~ 2 × 10
3individual/mL.
Further improvement to technique scheme: in described step (4), the concentration of spermatozoa diluent is 1 × 10
5~ 2 × 10
6individual/mL.
Compared with prior art, advantage of the present invention and good effect are: the present invention screens the suitableeest maturing agent and the suitableeest accelerating time that are applicable to starfish ovum by experiment, adopt this maturing agent can obtain rapidly the mature egg of high-quality, and operation will be waited to produce any impact on follow-up fertilization, thus establish a kind of method of quick acquisition starfish high-quality ovum and artificial insemination; And the present invention can not cause very major injury and dead to starfish parent, and parent can repeatedly use, and the acquisition of ovum is not subject to seasonal restrictions, and can accomplish to supply ovum as required, for carrying out biology of reproduction and genetics research provides good experiment material.
Accompanying drawing explanation
Fig. 1 is the starfish ovum form before and after embodiment of the present invention accelerating, and wherein Figure 1A is the sea star egg subgraph before accelerating, and Figure 1B is the sea star egg subgraph after accelerating.
Embodiment
Below in conjunction with the drawings and specific embodiments, technical scheme of the present invention is described in further detail.
Accelerating and the Artificial insemination procedures of starfish high-quality ovum of the present invention comprise the following steps:
1, the cultivation of starfish parent
20 of random selecting ripe starfish parents (determining the quantity of female parent and male parent according to sexual gland) are placed in cultivation in 100L aquarium, initial water temperature is 12 DEG C, adopts electrical bar to heat, heats up 0.5 ~ 1 DEG C every day, maintain this temperature to when 16 DEG C.Microzooplankton throw something and feed between culture period as halogen worm and Small Copepods, and throw something and feed 10 ~ 20g(every starfish, 0.5 ~ 1g at every turn), within 2-3 days, throw something and feed 1 time, changed water 100% every 6 days, parent culture is more than one week.
2, the collection of starfish gonadal tissue
The taper draw point of diameter 3mm is adopted to open an aperture gently on starfish one arm, a small amount of sexual gland is taken out (because starfish has 5 starfish arms with elbow tweezers, every bar starfish arm all has gonadal tissue, starfish also has very strong regeneration capacity simultaneously, only can't damage starfish from 1 ~ 2 starfish arm sexual gland that takes a morsel, starfish is by means of its rapid regeneration ability, original state can be reverted to rapidly, so it is very little to its biotic influence to gather a small amount of gonadal tissue, can not damage starfish individuality).Sexual gland is Chinese red to be female and to show that gonad development is good; The a small amount of female gonads taken out is put in the beaker that a little seawater is housed, is placed on ice, from female gonads, obtains the sea star egg liquid containing starfish ovum.Sexual gland is creamy white as male and show that gonad development is good, is put in 1.5mL centrifuge tube by a small amount of male gonad after taking out, shreds, get supernatant fluid and be starfish seminal fluid, be placed on ice with scissors.
3, the artificial ripening of starfish ovum
(in ovum liquid, the concentration of ovum is 1 × 10 to get 5mL sea star egg liquid
3~ 2 × 10
3individual/mL) be placed in the 50mL centrifuge tube that seawater is housed, add 1-methyl guanine and make its final concentration be 2 μMs, be placed in 16 DEG C of incubators, constantly shake with shaking table, maturation of ovum after 1h.Rinse ovum by fresh seawater, after flushing, ovum is placed in 50mL centrifuge tube, adopt fresh seawater washing ovum for subsequent use.Ovum in the present invention also can adopt existing immature ovum to carry out accelerating.
The present invention can have the function of accelerating starfish ovum through testing the creationary 1-of providing methyl guanine, and the usage amount of 1-methyl guanine also has vital effect, only have suitable consumption just can play the effect of effective accelerating starfish ovum, consumption is too high or too low does not all have accelerating effect.Fig. 1 uses the starfish ovum aspect graph before and after 1-methyl guanine accelerating, and can see accelerating from the microscopic examination photo of Figure 1A and Figure 1B before, ovum shows slightly irregular, has reproduction to steep in ovum, and the ovum after accelerating is rounded, and reproduction bubble disappears.
4, artificial insemination
Get 10 μ L starfish seminal fluid to be put in 1mL sea dilution with water and to obtain spermatozoa diluent (spermatozoa diluent after dilution is 5 × 10 through the about concentration of microscopic examination
5individual/mL), get spermatozoa diluent described in 300 μ L and join 5 × 10
3in ovum, shake gently, after 5 ~ 10 minutes, fertilization process completes, and adding up fertilization rate under anatomical lens is 95.8%, after being fertilized, and adopts fresh seawater to rinse fertilized egg, for subsequent use.
Above embodiment only in order to technical scheme of the present invention to be described, but not is limited; Although with reference to previous embodiment to invention has been detailed description, for the person of ordinary skill of the art, still can modify to the technical scheme described in previous embodiment, or equivalent replacement is carried out to wherein portion of techniques feature; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (8)
1. the accelerating of starfish high-quality ovum and an Artificial insemination procedures, is characterized in that it comprises the following steps:
(1) cultivation of starfish parent
Cultivate in aquarium starfish parent being placed in 15 ~ 16 DEG C of constant temperature, microzooplankton of throwing something and feeding between culture period, throws something and feeds once in 2 ~ 3 days, within 4 ~ 6 days, changes water 100%;
(2) collection of starfish gonadal tissue
Gather the ovum liquid in starfish arm female gonads and the seminal fluid in male gonad respectively;
(3) artificial ripening of starfish ovum
In the described ovum liquid collected, add the 1-methyl guanine that final concentration is 2 μMs, be placed in 16 DEG C of incubators, with maturation of ovum after shaking table shake 1 ~ 2h; Adopt fresh seawater washing ovum for subsequent use;
(4) artificial insemination
The described seminal fluid seawater dilution 50 ~ 100 times collected is obtained spermatozoa diluent, draws described spermatozoa diluent and join 2 × 10
3~ 5 × 10
3in individual ovum, then add 30 ~ 50mL seawater, shake 5 ~ 10 minutes gently, in dissection Microscopic observation fertilization effect, egg membrane lifts, and shows that fertilization completes.
2. the accelerating of starfish ovum according to claim 1 and Artificial insemination procedures, is characterized in that: in described step (1), initial water temperature is 12 DEG C, adopts electrical bar to heat, heat up 0.5 ~ 1 DEG C every day, maintain this temperature to when 16 DEG C.
3. the accelerating of starfish ovum according to claim 1 and Artificial insemination procedures, is characterized in that: described step (1) middle-size and small-size zooplankton is halogen worm and/or Small Copepods.
4. the accelerating of the starfish ovum according to claim 1 or 3 and Artificial insemination procedures, is characterized in that: the scale of feeding of described microzooplankton is every starfish 0.5 ~ 1g.
5. the accelerating of starfish ovum according to claim 1 and Artificial insemination procedures, it is characterized in that: adopting the taper draw point of diameter 2 ~ 3mm on starfish one arm, open a diameter gently in described step (2) is the aperture of 2 ~ 3mm, with elbow tweezers, well-developed a small amount of sexual gland is taken out, the female gonads of taking-up is put in a little seawater, be placed on ice, after being filtered by gauze, obtain starfish ovum; Shred centrifugal by the male gonad tissue of taking-up, getting supernatant fluid is seminal fluid, is placed on ice.
6. the accelerating of starfish ovum according to claim 5 and Artificial insemination procedures, is characterized in that: described sexual gland is Chinese red to be female and to show that gonad development is good; Sexual gland is creamy white as male and show that gonad development is good.
7. the accelerating of starfish ovum according to claim 1 and Artificial insemination procedures, is characterized in that: in described step (3), in ovum liquid, the concentration of ovum is 1 × 10
3~ 2 × 10
3individual/mL.
8. the accelerating of starfish ovum according to claim 1 and Artificial insemination procedures, is characterized in that: in described step (4), the concentration of spermatozoa diluent is 1 × 10
5~ 2 × 10
6individual/mL.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510178843.5A CN104798708B (en) | 2015-04-16 | 2015-04-16 | Maturity promoting and artificial fertilization method for high-quality ova of starfishes |
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|---|---|---|---|
| CN201510178843.5A CN104798708B (en) | 2015-04-16 | 2015-04-16 | Maturity promoting and artificial fertilization method for high-quality ova of starfishes |
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|---|---|
| CN104798708A true CN104798708A (en) | 2015-07-29 |
| CN104798708B CN104798708B (en) | 2017-03-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111296337A (en) * | 2020-03-24 | 2020-06-19 | 上海海洋大学 | Method for rapidly identifying ovum maturity of hyriopsis cumingii without damage |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070111077A (en) * | 2006-05-16 | 2007-11-21 | 한재명 | An apparatus and method for capturing starfish in the sea |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20070111077A (en) * | 2006-05-16 | 2007-11-21 | 한재명 | An apparatus and method for capturing starfish in the sea |
Non-Patent Citations (4)
| Title |
|---|
| ADIEYOU123: "受精的机制", 《百度文库＞教育专区＞高等教育＞理学HTTP://WENKU.BAIDU.COM/VIEW/DA44F7C32CC58BD63186BD31.HTML?FROM=SEARCH》 * |
| 桂丽萍等: "虾夷砂海星化学成分的研究", 《南开大学学报（自然科学版）》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111296337A (en) * | 2020-03-24 | 2020-06-19 | 上海海洋大学 | Method for rapidly identifying ovum maturity of hyriopsis cumingii without damage |
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