CN102440207B - Establishing method of Cnobilis inbred line - Google Patents
Establishing method of Cnobilis inbred line Download PDFInfo
- Publication number
- CN102440207B CN102440207B CN2011103036745A CN201110303674A CN102440207B CN 102440207 B CN102440207 B CN 102440207B CN 2011103036745 A CN2011103036745 A CN 2011103036745A CN 201110303674 A CN201110303674 A CN 201110303674A CN 102440207 B CN102440207 B CN 102440207B
- Authority
- CN
- China
- Prior art keywords
- ovum
- essence
- shellfish
- sperm
- individuality
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention relates to an establishing method of a Cnobilis inbred line, which can quickly purify the genome of the Cnobilis, so as to accelerate variety breeding. Individuals containing female and male germ cells and appearing as female, namely hermaphrodites, are selected through micrographic examination, an egg is fertilized by sperm in the same individual through induced parturition hastening or dissection fertilization, the amount of the sperms are increased or reduced artificially by taking measures to artificially interfere and adjust the relative proportion of the male and the female gametes in the water, and hatching, breeding and cultivating are carried out according to the ordinary technology of the species, so as to establish the inbred line. The establishing method of the Cnobilis inbred line is simple to operate, requires no special chemical reagent, and is easy to popularize and apply.
Description
Technical field
The invention belongs to the shellfish breeding technical field, be specifically related to a kind of method for building up of Cnobilis inbred line.
Background technology
Chlamys nobilis [Chlamys (Minachlamys) nobilis (Reeve, 1852)] is the water warm shellfish, and natural distribution is in Honshu, four countries, the nine divisions of China in remote antiquity of Japan, the ground such as the South Sea of China and Indonesia.In Guangdong Province coastal Haimen, hide the ground such as wave, Daya Gulf, Tai Pang Wan, lock slope distribution all arranged.Chlamys nobilis is gonochorism type ocean bivalve shellfish, reaches sexual maturity about 1 year, and female sexual gland is crocus, and male gonad is milky, and be the 5-10 month at Coast of Guangdong Province breeding period, take the 5-6 month as breeding period, ten thousand of the 300-1500 of can laying eggs.Extensively there are hermaphroditism and property conversion phenomena.The ratio of carrying out property conversion accounts for 4.47% of sum in anniversary, and wherein the male ratio of changing is 5.17%, and the female ratio of changing is 4.0%.The main time of origin of female, male consubstantiality and property conversion is in the 12-2 month and the 7-8 month.
At present, having in theory two kinds of methods to carry out the homozygotic structure of organism cultivates, what wherein isogeneity was fastest is by gynogenesis or androgenesis, but at present the androgenesis research of shellfish be there is no report, though the gynogenesis to shellfish has some researchs, but very large in the technical elements difficulty, still can not in practical study, use at present.Another method is inbreeding, the multiple mating strategy such as comprise selfing, full-sib mating, half-sib mating, backcross.Wherein, the nearest mating pattern of affiliation is selfing in the inbreeding, the isogeneity speed of selfing is far away faster than other inbreeding mode, selfing mainly is to utilize hermaphroditism or sex reversal phenomenon to set up inbred line, two kinds of methods can be arranged technically: the one, utilize sperm deepfreeze technology and property to turn phenomenon and set up inbred line in conjunction with carrying out selfing, but the sperm deepfreeze technical research of marine shellfish also is in initial stage at present, and the application of this technology also needs with time; Another kind of more feasible method is to utilize the ripe smart ovum of hermaphroditism individuality to set up inbred line by self-fertilization.Pacific oyster is a kind of gonochorism type shellfish, Hedgecock etc. (1995) utilize the female and male gametophyte of the hermaphroditism individuality of minority to carry out self-fertilization and have set up inbred line, Wang Meifang etc. (2006) are studied self-fertilization and the early embryonic development thereof of pteria martensii hermaphroditism individuality, find that autogamous fertilization rate is starkly lower than cross fertilization, there is more blastomere to disintegrate in the growth course, part embryo can develop into D type larva, but fails to cultivate seedling.Zhang Guofan etc. (2003) successfully utilize the bay scallop of hermaphroditism type to make up self-fertilization family (sff).For gonochorism type shellfish, the ratio of its female and male gametophyte consubstantiality individuality is lower, and it is female, the fully-developed situation is rare especially simultaneously for andro gamete.At Chlamys nobilis, although observed the existence of hermaphroditism by histologic section, the method for utilizing this phenomenon to make up inbred line is not reported, and is had no the report that carries out artificial insemination method by post-mortem method.
Summary of the invention
The object of the present invention is to provide a kind of method for building up of Cnobilis inbred line.
The present invention takes following scheme to achieve the above object: select hermaphroditic individuality from Chlamys nobilis, adopt the method for selfing to set up and cultivate inbred line.
The method of described selfing is: select gonad maturity, in appearance for female Chlamys nobilis, the microexamination gonadal tissue, pick out the individuality that contains simultaneously ovum and sperm, be that the hermaphroditism type is individual, it is carried out spawn-inducing, each individuality is independent discharge essence, ovum under the condition of mutually isolation, and essence, ovum that same individuality is produced carry out self-fertilization, then carry out embryo's hatching, larvae cultivation by the routine techniques of these species and form, in operating process, take conventional quarantine measures, avoid inbred line individuality in addition to sneak into wherein.
Its operating procedure is as follows:
The discriminating of hermaphroditism individuality: with sexual gland full, color is saffron shellfish numbering, each shellfish syringe needle puncture glandular tissue, spread upon and also drip seawater on the slide, observe placing microscopically on the slide, as containing ovum and sperm in the sample, should individuality be the hermaphroditism type then.
Hasten parturition and inseminate: after the hermaphroditism individuality of picking out was dried in the shade 30-60 minute, flowing water rushed 20-30 minute, and each shellfish is put into separately a container, added in advance the seawater than 5 ℃ of the high 3-of former cultivation water temperature in each container.Observe the Spawning situation of shellfish.Ovum and sperm that same individuality produces are fertilized, avoid other individual gametes to sneak into.
The concrete condition asynchronous for Spawning and quantity variance is greatly different, the present invention hasten parturition and fertilization process in take that following measure is artificial to be increased or reduce sperm quantity and carry out manual intervention and adjusting with the relative scale of essence, ovum in to water body: for the situation of laying eggs after producing essence first, when the water body that contains sperm presents milky, place another container to continue discharging the shellfish taking-up, and keep seminal fluid; When this shellfish begins to ovulate, at once this shellfish is taken out and flushing, then place fresh filtering sea to allow it lay eggs, when the quantity of ovum in the water body of laying eggs reach 10/more than the mL or when no longer discharging, take a sample and observe in microscopically, when the observable sperm quantity of ovum periphery below 50 % is less than one, 1:100 adds the seminal fluid of this shellfish by volume, and stirring water body, 3-takes a sample after 5 minutes and observes in microscopically, stops to add seminal fluid when the ovum periphery more than 90% can be observed 1-2 sperm; Change water with siphonage after 30 minutes.Produce afterwards the situation of essence for laying eggs first, when egg laying amount reach 10/when mL is above, shellfish taken out places another container allow its ejaculation, during then this shellfish of by volume 1:100 adding seminal fluid lays eggs.
Do not lay eggs or lay eggs and do not produce the concrete condition of essence for producing essence, the present invention adopts and dissects the sexual gland method and ammonia seawater stimulus method carries out artificial insemination.Do not lay eggs for producing essence, lay eggs and do not produce the individuality of essence, after routine is hastened parturition end, parent's gonad is dissected, then the gamete that obtains is carried out artificial insemination in the ammonia seawater.Concrete operation method is: cut the shellfish body open with scalpel, the clip sexual gland also shreds, and with 200-300 mesh sieve thin,tough silk bag extruding gonadal tissue, obtains extrudate.The situation of not laying eggs to producing essence is put into larger container with the gonadal tissue extrudate, fills in advance the ammonia seawater of 50-200 liters 0.002-0.005%, the water body on sucking-off container upper strata 2/3 after 30 minutes, and add filtering sea, repeat 3 times.Do not produce the situation of essence to laying eggs, then extrudate is placed the ammonia seawater of the 0.002-0.005% of 1L, volume ratio by 1:100 adds in the ovum of having collected, the quantity of sperm around the microexamination ovum, when the sperm around 50% ovum is less than 1, add seminal fluid until the ovum 90% or more stops when 1-2 sperm being arranged on every side.
Routinely technology hatch, larvae cultivation and cultivation, and prevent pollution between different parents.
Step crucial among the present invention is: the hermaphroditism that (1) utilizes Chlamys nobilis naturally to exist identifies the hermaphroditism individuality by micrography; (2) obtain the gamete that can not obtain by revulsion by post-mortem method, and promote essence, the maturation of ovum by ammonia seawater stimulus method, and finish fertilization and embryonic development; (3) sperm quantity is too much, can cause Chlamys nobilis embryonic development failure, the present invention to adopt the adding of the auxiliary control of the method sperm volume of microscopy, and the density by increasing water body and changing the method dilution sperm of water, adjusts the ratio of smart ovum.
Advantage of the present invention: the present invention utilizes the hermaphroditism special entity in the Chlamys nobilis of gonochorism type to set up inbred line, can accelerate the purifying speed of Chlamys nobilis gene, accelerates breed breeding speed.The present invention is easy and simple to handle, do not use special chemical reagent, easily applies.
Embodiment
In November, 2009
The sexual gland of picking out for two ages from the Tai Pang Wan cultured population was fuller, color is 70 of saffron Chlamys nobilis, in indoor culture 3 days.During storing up, change water every day once, 22 ℃ of water temperatures, and the sufficient bait of throwing something and feeding promote that sexual gland further develops into the physiological ripening state.Shellfish is arranged numbering, and each shellfish is with syringe needle puncture glandular tissue, spreads upon on the slide and drips seawater, will place microscopically to observe on the slide, find in the gonadal tissues of 4 shellfishes existing ovum again with sperm is arranged.Then stimulation by running water 30 minutes again after these 4 hermaphroditism type individualities were dried in the shade 60 minutes places shellfish respectively in the glass beaker that fills 1L seawater (26 ℃ heat up 4 ℃) of 4 numberings.
1,2, No. 4 individualities were about 10 minutes beginning ejaculations, when water body is creamy white, the shellfish taking-up is placed another beaker, and add same intensification seawater, after changing water 3 times, observe 1, No. 4 individuality and begin ovulation, shellfish is taken out and wash with filtering sea, then place the clean approximately 5L glass jar of 3L filtering sea that fills, after 10 minutes, collected a large amount of ovum, add simultaneously the seminal fluid 1ml that corresponding numbering shellfish produces, stir evenly water body with glass bar, the water body that takes a morsel is observed in microscopically, sperm moves about, oval; But the sperm quantity around No. 1 individual ovum is more, approximately has the sperm around 40% the ovum to reach more than 10, and only 1-2 of the sperm quantity around No. 4 individual ovum.Add up fertilization rate after 2 hours, the fertilization rate of 1, No. 4 individuality is respectively 30% and 78%.For Chlamys nobilis, smart archiblast amount, the maturity of producing except each individuality are different, they can cause outside the different fertilization rate, sperm quantity also is a most key factor, quantity too much can cause hatching unsuccessfully, No. 1 individual fertilization rate is lower, and possible main cause is that sperm quantity is too much.Generally speaking, it is individual more suitable that the sperm quantity around the ovum is controlled at 1-2, such as No. 4 individualities.Collect the floating larva after 10 hours and carry out the routine cultivation, No. 1 individuality after cultivating in 3 days, and larva is all dead; No. 4 individualities began to occur eyespot and dropped into collector in the time of the 13rd day, and larval density is 15/100ml.In March, 2010, No. 4 shellfish has 120 shellfish survivals, and size is 2.32cm.
No. 2 individuality began ovulation in 30 minutes, but quantity is few, the basic inertia of its sperm of microexamination, and avette attitude is irregular, abandons.
No. 3 individualities do not produce essence and lay eggs.
In May, 2011
Pick out from the Daya Gulf cultured population that 30 sexual glands are full, color is saffron Chlamys nobilis, in indoor culture 4 days, store up 25 ℃ of water temperatures.Shellfish is arranged numbering, and each shellfish is with syringe needle puncture glandular tissue, spreads upon on the slide and drips seawater, will place microscopically to observe on the slide, find in the gonadal tissue of 1 shellfish existing ovum again with sperm is arranged.Then stimulation by running water 20 minutes again after this hermaphroditism type individuality dried in the shade 30 minutes places shellfish in the glass jar that fills 3L seawater (29 ℃ heat up 4 ℃).Begin ejaculation after 15 minutes, still do not observe ovulation after 60 minutes, at once this is changed that shellfish is taken out and with cutting open behind the seawater flushing, with scissors clip sexual gland and shred this tissue, and place 200 purpose bolting silk mesh bags to push, the yellow liquid that squeezes out is placed 0.0025% the ammonia seawater of 100L, microscopy finds that sperm is too much, and most of ovum is transparent, the small part oval, suck 2/3 supernatant after 30 minutes, add again filtering sea, repeat 3 times after, the ovum that sinks to container bottom changed in the 10L container hatches, adding up fertilization rate after 2 hours, is 73%.Collect the floating larva after 8 hours, and technology is carried out larvae cultivation routinely, obtained afterwards the juvenile mollusk of 30 300-500um in 23 days.
Claims (4)
1. the method for building up of a Cnobilis inbred line is characterized in that: select hermaphroditic individuality from Chlamys nobilis, adopt the method for selfing to set up and cultivate inbred line; Obtain gamete by post-mortem method, and promote essence, the maturation of ovum by ammonia seawater stimulus method, finish fertilization and embryonic development;
The method of described selfing is: select gonad maturity, in appearance for female Chlamys nobilis, it is carried out spawn-inducing, each individuality is independent discharge essence, ovum under the condition of mutually isolation, and essence, ovum that same individuality is produced carry out self-fertilization, then carries out embryo's hatching, larvae cultivation and form; In operating process, take quarantine measures, avoid inbred line individuality in addition to sneak into wherein;
For producing first the situation of laying eggs after the essence, take following measures: when the water body that contains sperm presents milky, place another container to continue discharging the shellfish taking-up, and keep seminal fluid; When this shellfish begins to ovulate, at once this shellfish is taken out and flushing, then place fresh filtering sea to allow it lay eggs, when the quantity of ovum in the water body of laying eggs reach 10/more than the mL or when no longer discharging, take a sample and observe in microscopically, when the observable sperm quantity of ovum periphery below 50 % is less than one, 1:100 adds the seminal fluid of this shellfish by volume, and stirring water body, 3-takes a sample after 5 minutes and observes in microscopically, stops to add seminal fluid when the ovum periphery more than 90% can be observed 1-2 sperm;
Produce afterwards the situation of essence for laying eggs first, take following measures: when egg laying amount reach 10/when mL is above, shellfish taken out places another container allow its ejaculation, during then by volume 1:100 adding seminal fluid is laid eggs to this shellfish;
Do not lay eggs or lay eggs and do not produce the situation of essence for producing essence, take following measures: do not lay eggs for producing essence, lay eggs and do not produce the individuality of essence, hasten parturition in routine and parent's gonad is dissected after finishing, then the gamete that obtains is carried out artificial insemination in the ammonia seawater.
2. the method for building up of described Cnobilis inbred line according to claim 1, it is characterized in that: the discrimination method of hermaphroditism individuality is: with syringe needle puncture glandular tissue, spread upon and also drip seawater on the slide, observe placing microscopically on the slide, as containing ovum and sperm in the sample, should individuality be that hermaphroditism is individual then.
3. the method for building up of described Cnobilis inbred line according to claim 1, it is characterized in that: hasten parturition and the method for inseminating is: after the hermaphroditism individuality of picking out was dried in the shade 30-60 minute, flowing water rushed 20-30 minute, each shellfish is put into separately a container, add in advance the seawater than 5 ℃ of the high 3-of former cultivation water temperature in each container; Observe the Spawning situation of shellfish; Ovum and sperm that same individuality produces are fertilized, avoid other individual gametes to sneak into.
4. the method for building up of described Cnobilis inbred line according to claim 1, it is characterized in that: do not lay eggs or lay eggs and do not produce the situation of essence for producing essence, take following measures: cut the shellfish body open with scalpel, the clip sexual gland also shreds, with 200-300 mesh sieve thin,tough silk bag extruding gonadal tissue, obtain extrudate; The situation of not laying eggs to producing essence is put into larger container with the gonadal tissue extrudate, fills in advance the ammonia seawater of 50-200 liters 0.002-0.005%, the water body on sucking-off container upper strata 2/3 after 30 minutes, and add filtering sea, repeat 3 times; Do not produce the situation of essence to laying eggs, then extrudate is placed the ammonia seawater of the 0.002-0.005% of 1L, volume ratio by 1:100 adds in the ovum of having collected, the quantity of sperm around the microexamination ovum, when the sperm around 50% ovum is less than 1, add seminal fluid until the ovum 90% or more stops when 1-2 sperm being arranged on every side.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103036745A CN102440207B (en) | 2011-10-10 | 2011-10-10 | Establishing method of Cnobilis inbred line |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011103036745A CN102440207B (en) | 2011-10-10 | 2011-10-10 | Establishing method of Cnobilis inbred line |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102440207A CN102440207A (en) | 2012-05-09 |
| CN102440207B true CN102440207B (en) | 2013-10-23 |
Family
ID=46003535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011103036745A Active CN102440207B (en) | 2011-10-10 | 2011-10-10 | Establishing method of Cnobilis inbred line |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102440207B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102960276B (en) * | 2012-11-20 | 2014-02-12 | 汕头大学 | Cultivation method of Chlamys nobilis new strain enriched with DNA (Deoxyribonucleic Acid) and application thereof |
| CN103828746B (en) * | 2014-03-04 | 2016-01-20 | 大连海洋大学 | A kind of mating system of Ruditapes philippinarum |
| CN115413612A (en) * | 2022-09-15 | 2022-12-02 | 中国科学院南海海洋研究所 | Non-irritant gathering and spawning method for holothuria leucospilota |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1739343A (en) * | 2004-08-24 | 2006-03-01 | 中国科学院海洋研究所 | A kind of tetraploid preparation method who is applicable to chlamys farreri |
| CN1263374C (en) * | 2004-10-10 | 2006-07-12 | 中国科学院海洋研究所 | Method for setting up dioecism type mussel inbred line |
| CN101341862B (en) * | 2008-08-29 | 2010-12-15 | 中国科学院南海海洋研究所 | Fry production method for Chlamys nobilis with yellow shell |
-
2011
- 2011-10-10 CN CN2011103036745A patent/CN102440207B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102440207A (en) | 2012-05-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103798167B (en) | The artificial breeding method of a kind of Ostrea cucullata | |
| CN101703016B (en) | Technology for culturing unisexual hybrid scallop by utilizing purple scallop and bay scallop | |
| CN102301970B (en) | Method for cultivating hybridized and backcrossed Argopecten product fries | |
| CN101796927B (en) | Artificial breeding method of coregonus paled | |
| CN102939924A (en) | Method for artificially breeding sepia lycidas gray | |
| CN103798166A (en) | Method for large-scale indoor artificial breeding of Hong Kong oysters in coastal region of South China | |
| CN103461253B (en) | Yellow River fish artificial fecundation method | |
| CN100362912C (en) | Method for making seed of Pinctada martensii of red shell strain | |
| CN101720691A (en) | Seed production method for breeding pinctada martensii | |
| CN103210852A (en) | Method for obviously improving survival rate of exopalaemon carinicauda in artificial breeding process | |
| CN1372790A (en) | Artificially cultured triploid Hepu nacre and its pearl culturing method | |
| CN102440207B (en) | Establishing method of Cnobilis inbred line | |
| CN103828746B (en) | A kind of mating system of Ruditapes philippinarum | |
| CN101341862B (en) | Fry production method for Chlamys nobilis with yellow shell | |
| CN104381158B (en) | Method for breeding larvae-type oysters by oviparous-type means | |
| CN101743915B (en) | Cultivation method for enhancing physique of weak young Chinese sturgeon | |
| CN102630622A (en) | Artificial breeding method of Bostrichthys sinensis | |
| CN101278659A (en) | Method for obtaining variety of Oplopanax elatus with heterosis | |
| CN102835356B (en) | Breeding method of artificial parents of sipunculus nudus | |
| CN106614123B (en) | A kind of pressure shock induction great flounder subtrahend gynogenesis method | |
| CN104430067B (en) | A kind of propagation method of Monopterus albus (Zuiew) | |
| CN103109765B (en) | Seed production method by interspecific crossing of green abalone and Pacific abalone | |
| CN104938263A (en) | Method for controlling greenhouse vegetable aphids through adoption of harmonia axyridis | |
| CN102119674B (en) | Cynoglossus semilaevis triploid fry mass inducing method | |
| CN105815246A (en) | Hybrid seed production method for Argopecten irradians concentricus (female) and Chlamys nobilis (male) |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |