CN1263374C - Method for setting up dioecism type mussel inbred line - Google Patents
Method for setting up dioecism type mussel inbred line Download PDFInfo
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- CN1263374C CN1263374C CN200410050568.0A CN200410050568A CN1263374C CN 1263374 C CN1263374 C CN 1263374C CN 200410050568 A CN200410050568 A CN 200410050568A CN 1263374 C CN1263374 C CN 1263374C
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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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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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
- A01K2227/00—Animals characterised by species
- A01K2227/70—Invertebrates
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a technique for producing shellfish seeds, and particularly to provides a method for establishing a dioecism type shellfish inbred line. A special hermaphroditic individual which simultaneously has two paroecious germ cells is selected from a dioecism type shellfish, and adopts a selfed strategy to establish and culture an inbred line. The genital color of the dioecism type shellfish can be observed by naked eyes between the female and the male of the same specie under the in vivo condition, and thus, the sexual difference can be discriminated. The present invention can utilize a special variation individual in the dioecism type shellfish to establish the inbred line, accelerate the gene purification speed of the dioecism type shellfish, quickly reject the recessive lethal genes, and greatly accelerate the variety breeding speed of the dioecism type shellfish. The present invention is easy to operate, has no need of using particular chemical reagents, has no need of particular devices, and is easy to popularize and apply. The present invention can be used for directly identifying and determining the hermaphroditic individual of the dioecism type shellfish through the visual observation under the in vivo condition.
Description
Technical field
The present invention relates to the seeding technique of shellfish, a kind of method for building up of gonochorism type shellfish inbred line is provided especially.
Background technology
Shellfish mostly is the gonochorism type, and a few species is the hermaphroditism type, and the sex that some kinds are also arranged is unsettled.Gonochorism type shellfish shows as gonochorism on the species level, be next individual one type the gonad that only has in female or male of normal condition, after gonad is reached maturity one by one body only can produce a kind of gamete in female (ovum) or male (sperm); Hermaphroditism type shellfish shows as hermaphroditism on the species level, being each individuality all has female and male etc. two types gonad simultaneously in the gonad developmental stage, and each individuality can produce female and male two kinds of gametes when gonad was reached maturity.
In lamellibranchiata, a few species such as the big scallop during only scallop belongs to, bay scallop are hermaphroditism type species, most shellfishes in the lamellibranchiata are the gonochorism type, are gonochorism type shellfish as stalwart blood clam, mussel, Patinopecten yessoensis, chlamys farreri, long oyster, Ruditapes philippinarum etc.Hermaphroditism type shellfish is more relatively in the Gastropoda, but row most of kinds in vitro fertilization also are gonochorism type animals, are the gonochorism type as all Bao Ke animals.
Two kinds of gonad of male and female can take place in the gonochorism type shellfish once in a while to be organized and comes across the phenomenon of body one by one simultaneously, promptly hermaphroditic phenomenon.Be embodied in when gonad is reached maturity, existing male reproductive cell also has female reproductive cell in the gonad of same individuality.The probability that occurs above-mentioned hermaphroditic special entity in gonochorism type shellfish has bigger difference according to the species difference: it is higher relatively that wherein hermaphroditic probability appears in oyster, mussel, pearl shell, then rarely found in other gonochorism type shellfishes, though in species such as haliotis discus hannai Ino, comb pen shell, Patinopecten yessoensis, chlamys farreri, Coelomactra antiquata, paphia undulata report is arranged, hermaphroditism is all discussed as the improper special case of corresponding species.Simultaneously, the individuality of all these tool hermaphroditisms of finding in gonochorism type shellfish all is observed by Histological method after putting to death research object.In all gonochorism type shellfishes, there is no at present the report of under the live body situation, finding the hermaphroditism individuality.
At present, have two kinds of methods to can be used for biological homozygotic quickly breeding in theory: what wherein isogeneity speed was the fastest is gynogenesis or androgenesis, only need a generation just can reach genomic high homogenous, but at present the androgenesis research of shellfish still there is not report, though the gynogenesis to shellfish has some researchs, but very big in the technical elements difficulty, still can not in practical study, use at present.
Another scheme is inbreeding, 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, and biological every selfing generation can make the purifying speed of target gene reach 50%, only needs six generations of selfing just can make the homozygosity of target gene reach 0.984.The isogeneity speed of selfing is far away faster than other inbreeding mode, thus the selfing technology can realize gene fast purifying, in biological breeding, have important effect.Yet by existing technology, selfing can only be applied to hermaphroditism type biology.
Consider that marine shellfish is a unicellular lower eukaryote, even if special circumstances such as sex reversal or hermaphroditism also may take place in the shellfish of gonochorism type, so might be in the hermaphroditism special entity of these species with the application of policies of selfing.
In the gonochorism type shellfish of sex reversal may be taken place, it was attemptable a kind of scheme that the scheme of the freezing preservation of use gamete (sperm or egg mother cell) is set up inbred line.Promptly when certain individuality reaches sexual maturity, it is hastened parturition and uses the gamete Refrigeration Technique to preserve its gamete, then this individuality is proceeded to cultivate, work as abiogenesis, or carry out inducing of physics or chemistry by artificial method and make it become the another kind of sex that is different from gamete sex when freezing and when reaching sexual maturity, then reach out for its gamete (egg mother cell or sperm) once more, make the gamete fertilization of the another kind of sex of the gamete of new acquisition and freezing preservation, then can set up inbred line, in this scheme, need to use freezing preservation of gamete and deforst technique, and the sex reversal of artificial orientation control also do not realize at present, still needs and will solve considerable technical barrier so implement this scheme.
In gonochorism type shellfish, if can under the live body situation, find hermaphroditic special entity in advance, then can be by planned artificial propagation and cultivation scheme, make same parent's female, andro gamete be fertilized and set up and cultivate inbred line, the key of implementing this scheme is to find hermaphroditic special entity in these species in advance under the live body situation.Because except that part oyster and pearl shell, remaining shellfish has only the ovum that process obtained of laying eggs under condition of living body by the parent that vigor is just arranged, and ability and normal development with sperm of the same race fertilization are just arranged; In other words the ovum that most of shellfishes obtain by the method for dissecting does not have the ability of fertilization or normal development.Therefore the hermaphroditism individuality of only finding under the live body situation just might be realized self-fertilization and set up inbred line.But, in all gonochorism type shellfishes, there is no the report of under the live body situation, finding the hermaphroditism individuality at present.
Haliotis discus hannai Ino, Patinopecten yessoensis and chlamys farreri etc. all are typical gonochorism type shellfishes, still do not have the report of sex reverse, also do not find the report of hermaphroditism live body.By BA and ASFA retrieval, in haliotis discus hannai Ino, only China Liu Yong peak etc. is reported in nineteen eighty-two and finds a routine hermaphroditism individuality; In chlamys farreri, only report such as China Liao Cheng justice is found a hermaphroditic individuality from 241 samples; In Patinopecten yessoensis, the ball nation justice of Japan etc. has also been found hermaphroditism.The hermaphroditism that above-mentioned three species are reported all fails gonadal outward appearance is being carried out perusal and finding, all is to be to find in the Histological research that carries out of means and confirm with the paraffin section.
Under the normal condition, only have a kind of in female or the arrenotoky gland and fully-developed gonad uniform hue, soft in each individuality of gonochorism type shellfish.There is very significant, macroscopic color distortion in the gonad of most of gonochorism type shellfishes between the individuality of female and male two kinds of different sexes, after gonad is grown to a certain degree from the naked eye be in appearance distinguishable its for female or male.The gonad of shellfish male is generally milky, and the gonad of female individuals then color is more.Such as presenting colors such as Chinese red, orange-yellow, maroon, pink according to different plant species behind the female reproduction gland maturity of gonochorism kind in the mussel order of Bivalvia, the pearl shell suborder, in the Bao Ke animal of other species such as Gastropoda, female reproduction gland such as haliotis discus hannai Ino is a puce for female reproduction gland blackish green, haliotis diversicolor Reeve then, and the arrenotoky gland of above-mentioned shellfish all is a milky.
The present patent application people is at haliotis discus hannai Ino in recent years, found the individuality that gonad is unusual in Patinopecten yessoensis and the chlamys farreri successively, its gonad form is normal, outward appearance is full, soft, but special character is gonadal irregular colour one, the gonad surface that is same individuality has female and male two kinds of gonadal colors of these species simultaneously, this phenomenon is significantly different with the gonad in the dioecious normal individual of above-mentioned species, the present patent application people finds that after deliberation the unusual individuality of above-mentioned gonad is hermaphroditic special entity, and this is the hermaphroditic live body that obtains first after by naked eyes gonadal outward appearance being observed in above-mentioned three species.But not finding has the people to utilize the hermaphroditic live body that is obtained in the above-mentioned species to set up inbred line, and the present patent application people is after research for many years, and successfully the method for setting up inbred line in this part gonochorism type shellfish is finished in research and development finally.
Summary of the invention
The object of the present invention is to provide a kind of method of in gonochorism type shellfish, setting up inbred line.
Technical scheme of the present invention is as follows:
The strategy that the present invention sets up gonochorism type shellfish inbred line is: from the shellfish of gonochorism type, select have simultaneously two kinds of reproductive cell of male and female, be hermaphroditic special entity, adopt the strategy of selfing to set up and cultivate inbred line;
Wherein: described gonochorism type shellfish refer to its gonadal color or form same species female and male between have the difference that can under the live body situation, differentiate its sex by perusal;
After special entity in the described gonochorism type shellfish that has two kinds of reproductive cell of male and female simultaneously refers to that its gonad is grown, can recognize the gonad that this individuality has female, male two kinds of colors or form simultaneously by naked eyes in appearance from gonadal.By observation to the gonad surface color, selection is used as parent of the present invention based on female reproduction gland, arrenotoky gland area small individuals: with surface area, the ratio that the arrenotoky gland accounts for the gonad gross area less than, equal 30%, in perusal, as seen have the arrenotoky gland at least and get final product.The used hermaphroditic parent of the present invention screens by the following method: the gonad gonochorism type shellfish was grown to the maturing stage, check the gonad of each shellfish one by one, examine the color on gonad surface, if female and male two kinds of gonadal colors of these species appear in the gonad surface of body simultaneously one by one, determine tentatively that then this individuality is hermaphroditic special entity, should individuality take out and cultivate separately, by the gonad developmental state hermaphroditism individuality of selecting is continued to cultivate after gonad is reached maturity and be used to set up inbred line.Above-mentioned parent with hermaphroditism feature can search from life, without artificial induction's processing naturally shellfish (comprising the shellfish that the sea area is wild and propagate artificially), also can induce it to produce hermaphroditism by artificial physical or chemical treatment.
The strategy of described employing selfing is set up and is cultivated inbred line and be meant that the female and male gametophyte of each parent independently being hastened parturition and same parent being produced carries out self-fertilization under the condition of isolating mutually, carry out embryo's hatching, larvae cultivation by the routine techniques of these species then 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:
Methods such as employing dry in the shade stimulation, thermal stimulus and ultraviolet ray stimulation are hastened parturition: after the parent was dried in the shade 20～40 minutes each parent is put into a container separately and hasten parturition, add 3～5 ℃ of former cultivation water temperature raisings than the parent, the filtering sea that the process ultraviolet irradiation is handled in each container in advance.The close observation parent discharges the situation of female and male gametophyte, if emission performance product not as yet in 30 minutes after parent's entry was changed the seawater of hastening parturition for 1 time in then per about 30 minutes, discharges until sexual product.Make same parent's ovum and sperm fertilization, avoid other parents' gamete to sneak into.
At the concrete condition of two kinds of gonadal relative scales of male and female difference great disparity between different parents, 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 scales of female, male two kinds of gametes in to water body: low excessively parent takes Topographic method to increase sperm in the water body to improve fertilization rate after normally hastening parturition for arrenotoky gland ratio; Otherwise, then inject serotonin for the excessive parent of arrenotoky gland ratio and impel most sperm to excrete the sperm of discharging synchronously with ovum in advance to reduce.Be specially:
The situation that male sex-cell is very few: if one by one the arrenotoky gland surface area of body less than, equal 0.5% of this individual reproduction gland surface area summation, then the relative area of arrenotoky gland is too small, when hastening parturition the discharging the sperm total amount less.Use microscopic examination according to the conventional method back of hastening parturition, if do not observed sperm attached to outside the egg membrane around surpassing 10% egg mother cell, ovum sperm quantity deficiency on every side then.When confirming that sperm quantity is not enough, hasten parturition in routine and to finish the back topography is carried out at position, parent's arrenotoky gland place, will join this individuality then by the seminal fluid that anatomic method obtains in right amount and hasten parturition in the property product of normal discharging by routine: with fresh seawater with sperm dilution extremely lighter milky (every milliliter contains sperm 10
7～10
8Individual), divide then and get in the water body that 10～50 milliliters of dilution seminal fluid join the ovum that contains identical parent and fertilized egg for several times, at every turn, the careful synchronously water body that stirs when adding seminal fluid, take a sample after 3～5 minutes and observe, can be observed to the ovum periphery more than 98% and stop to add seminal fluid when having a sperm at least in microscopically.This method is equally applicable to the parent of sperm quantity deficiency in the less so that water body of some discharge capacity of sperm when hastening parturition.
The situation that male sex-cell is too much: the surface area of arrenotoky gland surpasses the individuality of this individual reproduction gland surface area summation 15%, be that arrenotoky gland relative area is excessive, sperm after hastening parturition in the water body will be seriously excessive, microscopically is observed number of sperm around each egg mother cell on the fixed focal length plane can reach dozens of even up to a hundred, must reduce the sperm in the water body in this case.The present invention adopts the method for injection serotonin to solve the excessive problem of sperm: plant shellfish injection serotonin solution 100～200 μ l to each behind parent's dry in the shade stimulation, serotonin solution is prepared with the aseptic seawater of the filtering with microporous membrane of via hole diameter 0.22 μ, and concentration is 20～500 μ mol/L.Hasten parturition in the ultraviolet irradiation seawater than 3～5 ℃ of former cultivation water temperature raisings then, the parent begins to discharge sperm in entry about 20～40 minutes, but does not discharge ovum in this stage.With each parent's seminal fluid respectively with beaker keep 200～500 milliliters standby, with fresh seawater dilution activation, abandon remaining seminal fluid and parent and the container of hastening parturition changed water and flushing repeatedly to remove sperm as far as possible during use.The parent has the process that is the pulse type emission ovum for 2～4 times in 2.5～9 hours after entry, have sperm to discharge simultaneously but quantity significantly reduces.Collect the property product that discharges rapidly behind the discharge ovum at every turn, observe ovum sperm quantity on every side immediately, if the sperm quantity deficiency in the water body can add the dilution of the seminal fluid of reservation in advance in batches; When surpassing 10～12, the sperm quantity of a focal plane around average each egg mother cell of microscopically must dilute or wash ovum immediately with a large amount of clean seawater.Surface area for the arrenotoky gland is the individuality of this individual reproduction gland surface area summation 10～15%, then adopts the method for above-mentioned injection serotonin can obtain than the better effect of the method for hastening parturition naturally, can significantly reduce ovum sperm quantity on every side.
Except that above-mentioned two kinds of solutions, also can be according to female and ratio situation arrenotoky gland surface area, ovum that measures such as the water body of hastening parturition and being fertilized by control, fertilization time are regulated each parent among a small circle and produced and the relative scale between sperm, in time with the sperm in the microscopic examination water body, as far as possible with one fixedly on the focal plane each egg mother cell number of sperm on every side be controlled in 10.Its basic principle is as follows: when white portion (arrenotoky gland) area on gonad surface less (surface area of arrenotoky gland account for this individual reproduction gland surface area summation 3% in), then take less water body (10～20 liters) to hasten parturition, be fertilized, and use long fertilization time (20～40 minutes); The surface area of white portion big (surface area of arrenotoky gland surpass this individual reproduction gland surface area summation 6%) then adopts and dilutes as early as possible after the water body of hastening parturition and be fertilized, the gamete of big (60～150 liters) are discharged or wash ovum.
The present invention has following advantage:
The present invention utilizes the special variation individuality in the gonochorism type shellfish to set up inbred line, can accelerate the isogeneity speed of gonochorism type shellfish, eliminates recessive lethal gene fast, accelerates the breed breeding speed of gonochorism type shellfish greatly.The present invention is easy and simple to handle, do not use special chemical reagent, do not need special facility, applies easily.The present invention can be applicable to that all can confirm as hermaphroditic individuality by the perusal Direct Recognition in the gonochorism type shellfish under the live body situation.
Embodiment
Below in conjunction with embodiment the present invention is further detailed.
Embodiment 1
Patinopecten yessoensis is typical gonochorism type shellfish, and its gonad is the ox horn shape after growing, and the female reproduction gland is Chinese red, malely then is milky that the sex of Patinopecten yessoensis is differentiated from gonadal being easy in appearance.
Culture 12230 Patinopecten yessoensis to marine site, Changhai County, Dalian gonad fully-developed and investigate certain year February, therefrom found 81 Patinopecten yessoensis with hermaphroditism feature.High 10～the 13cm of these Patinopecten yessoensis shells and have a common feature: the zone that promptly all has Chinese red and two kinds of colors of milky in each individual gonad simultaneously, wherein the surface area sum in milky zone shared rate variable scope in gonad surface area summation is very big, do not wait about from 0.1% to 80%, the gonad of two kinds of colors is organized phase embedding each other, but boundary is clear, clearly demarcated between two kinds of colors.Select the different individuality of arrenotoky gland surface area ratio to carry out breeding operations such as artificial induced spawning, insemination, cultivation for the parent, specific operation process is as follows:
1) parent supports temporarily
Above-mentioned shell surface with Patinopecten yessoensis of hermaphroditism feature is cleared up, carefully removed the attachment on shell surface and wash the back and supported 3 hours temporarily, set up inbred line the same day in 5 ℃ of water temperatures (natural temperature).
2) parent selects
From the hermaphroditism Patinopecten yessoensis of gonadal maturation, choose ten individualities according to arrenotoky gland (milky zone) ratio difference and hasten parturition and self-fertilization as the parent, set up ten inbred line: H
1, H
2, H
3, H
4, H
5, H
6, H
7, H
8, H
9, H
10H wherein
10Proportion maximum in milky zone reaches 50% in parent's gonad of self-fertilization family (sff), and promptly half zone, gonad surface is a milky, and remainder be a redness; H
2The parent takes second place, and the milky area accounts for 15% of gonad area summation; Milky zone proportion is lower in all the other parent's gonad, all is lower than 3% (seeing Table 1).H
10And H
2Parent's milky zone intersperses among whole gonadal each position, other eight parents' spermary distributes, and a common feature is then arranged is that the milky zone mainly is distributed in scallop near in the gonad at ventral margin position, promptly is distributed in the most advanced and sophisticated of gonad horn shape portion or near nearby the lateral margin position tip.
3) hasten parturition, be fertilized, hatch
With the shell surface of filtering sea outwash kind shellfish, the 30 fens kinds of drying in the shade at ambient temperature, each parent puts into separately in one 20 liters the Plastic Drum and hastens parturition, the distance between each barrel remains on more than one meter.Add 15 liters in the bucket in advance, be preheated to 10 ℃ ultraviolet irradiation filtering sea, 1 ultraviolet intensification seawater of replacing in 30 minutes after entry.Keep a close eye on the gamete emission behaviour after parent's entry, especially note observing and writing down the discharge capacity of sperm, in time take measures according to the sperm in the water body.In one hour, all ten parents all discharge gamete in a large number after the entry, all contain ovum (egg mother cell) and sperm simultaneously in the gamete of each parent's discharging, and ovum very fast sperm with same individual after discharge is fertilized.Microscopically is observed all ten reproductive cell that the hermaphroditism Patinopecten yessoensis is discharged, gamete with the gonochorism Patinopecten yessoensis does not have significant difference in appearance, strong, the active motion of sperm viability, and the yolk particle is greatly and obviously in the ovum cytoplasm, be evenly distributed the about 75 μ m of egg cell diameter.
Wherein, remove H
10And H
2Sperm concentration is lower in eight outer parents the property product, and the number of sperm around each ovum is less than 5, and therefore operation is simpler, promptly finishes to hasten parturition behind 2 subpulse formulas discharging ovum, the parent is taken out get final product from bucket.The property product of discharging was kept in the water body of hastening parturition 40 minutes, during careful stir 5 times so that ovum is in suspended state and have sufficient chance to contact with sperm.Hatch cultivation after then fertilized egg directly being diluted with fresh seawater, 8 ℃ of water temperatures of hatching, density are controlled at 40 every milliliter, and measures such as ovum are not taked to wash in the centre.
For H
10And H
2Deng two bigger parents of arrenotoky gland area ratio, take some following measures when hastening parturition: utilize the gamete of shellfish to be the characteristics of pulse type emission, each emission product is collected in batches, handled, after each gamete discharging, immediately emission product is transferred in 120 liters of clean drums of carrying out mark in advance and diluted with fresh seawater, after forming fertilization membrane, wash ovum immediately, make it continue discharging with the intensification seawater that adds ultraviolet irradiation behind the fresh seawater flushing parent and the container of hastening parturition again simultaneously to remove sperm unnecessary in the water body.Each parent's of Care Mark emission product is looked the fertilization situation and is determined abandoning of each batch fertilized egg to stay, and each batch ovum that same parent produced that will keep at last merges.Wherein collect H altogether
2Five batches of emission product of parent, first and third batch of property product number of sperm around average each egg mother cell on the visible focal plane of microscopically is respectively 12 and 15, second, four, five batch average number of sperm then reaches 20,35 and 31.Collect sperm quantity wherein less relatively first and emission product for the third time, be diluted to 40 every milliliter after the merging and hatch cultivation.H
10The parent has discharged three times altogether; each time emission product all mainly is a sperm, and each discharging back water body all is creamy white, and sperm concentration is big especially; it is very active to move; ovum negligible amounts, the ovum summation of three dischargings are less than 90,000, and visible each egg mother cell of microscopically is all closely surrounded by a large amount of sperms; each fixedly the number of sperm on the focal plane all reach more than hundred; sperm is lined up three layers outside egg mother cell, all ovums all make the molten mistake of egg membrane too much because of sperm, can't add up fertilization rate.After washing ovum, merge three emission product, under 40 every milliliter density, hatch and cultivate back H
10Family is not examined D shape larva, illustrates that the excessive then sperm of the area output of milky in the gonad (spermary) part is too much, and ovum can't normally be hatched.Wherein with H
10Sperm to a dioecious normal female Patinopecten yessoensis F
1Ovum carry out artificial insemination, then fertilization rate and incubation rate are respectively 100% and 85.28% (table 1), and H is described
10Parent's sperm has the ability of normal fertilization and hatching, i.e. H
10The gamete that produces is normal on function.
Each self-fertilization family (sff) obtains the ovum amount between 120～4,000,000, and when development of fertilized ova to two cell, the statistics fertilization rate the results are shown in Table 1, wherein H
10The fertilization rate of family can't be added up, incubation rate is zero, H
2The fertilization rate of family and incubation rate are respectively 68.91% and 24.26%, and the fertilization rate of all the other eight familys is added up incubation rate between 5.86%～76.25% in D shape larval stage between 92.4%～100%.
4) larvae cultivation, collect seedling and juvenile mollusk is cultivated
After embryonic development is floated to D shape larva, select with 200 mesh sieve thin,tough silk.Cultivate every milliliter of 8 larvas of density, cultivate 15 ℃ of water temperatures, each family breeding condition is identical.Make open-mouthed bait with chrysophyceae, cultivate to add after five days and throw diatom, day bait throwing in 4 times when feeding bait, gently with the bait input, is avoided cultivating water body and is spilt.Every day, full dose was changed water once, fell the pond once in per 5 days, was changing among the water process, and the isolation work between each family is carried out in strictness, avoids the larva of other familys to sneak into.When eyespot appears in 40% larva, throw in the polyethylene lace curtaining and collect seedling as adherance.Juvenile mollusk after the metamorphosis increases daily ration, feeding quantity according to the situation of ingesting, and every day, partly amount and full dose were changed water once respectively.The 10th day begins to observe quantity and the growth rate of measuring juvenile mollusk, self-fertilization family (sff) H as a result from the lace curtaining sampling after metamorphosis
1～H
9All obtained the juvenile mollusk of some, the juvenile mollusk number that the throwing adherance was added up in the time of 30 days sees Table 1.
The cultivation parameter of each self-fertilization family (sff) of table 1 Patinopecten yessoensis
| Family | Milky region area (%) | Ovum amount (ten thousand) | Fertilization rate (%) | Incubation rate (%) | Juvenile mollusk number (individual) |
| H 1 H 2 H 3 H 4 H 5 H 6 H 7 H 8 H 9 H 10 F 1×H 10 P | 0.7 15 0.8 0.9 0.6 0.8 2.6 0.8 1.5 50 / / | 220 120 260 350 180 260 400 360 210 8.7 300 500 | 94.40 68.91 93.47 96.67 92.42 100.0 95.83 95.67 100.0 / 100.0 92.12 | 76.25 24.26 73.19 36.23 13.33 29.03 11.74 5.86 21.74 0 85.28 40.25 | 24000 8600 14100 86000 7900 20900 16000 3800 11300 / / 182000 |
Annotate: the P group is to carry out the control group that colony hastens parturition and artificial insemination is set up with dioecious, gonad normal appearance Patinopecten yessoensis; Juvenile mollusk quantity is to throw the data of adding up in 30 days behind the adherance.
Above-mentioned multiply test proves, the Patinopecten yessoensis that all gonad surfaces have Chinese red and two kinds of color regions of milky simultaneously is hermaphroditism, and the gamete that all hermaphroditic Patinopecten yessoensis discharged all has the ability of normal fertilization and growth, be that its sperm and ovum are all normal on function, prove that hermaphroditic Patinopecten yessoensis can set up and cultivate self-fertilization family (sff) by the approach of selfing.
Embodiment 2
After Patinopecten yessoensis tool hermaphroditism feature, that the milky region area is bigger is fetched from the sea, the attachment on careful removal shell surface also washes, supported 12 days temporarily with sand filter seawater, the unicellular algas such as Phaeodactylum tricornutum of throwing something and feeding during this time are bait, change water every day twice, each 1/2, full dose was changed water once in per three days, supported 6 ℃ of water temperatures temporarily.
H
2, H
23, H
24, H
10Be the bigger hermaphroditic Patinopecten yessoensis of milky region area, the ratio that its milky region list area accounts for gonad surface area sum is respectively 15%, 20%, 30%, 50%, H wherein
2And H
10Identical with embodiment 1, standby through accelerating cultivation back, cultivate 6 ℃ of water temperatures.The operating process of setting up self-fertilization family (sff) is substantially the same manner as Example 1, its main difference is that present embodiment injects serotonin to the parent and emit prior to ovum to impel a large amount of sperms, so effectively reduce the sperm quantity in the water body, solved the too much problem of sperm quantity in the water body preferably.Specific operation process is as follows:
Drying in the shade handle 30 minutes after, respectively to serotonin 100～200 μ l (seeing Table 2) of above-mentioned four dioecious Patinopecten yessoensis injection 20～500 μ mol/L, in being warming up to 10 ℃ ultraviolet irradiation seawater, hasten parturition then, the container of hastening parturition is 60 premium on currency buckets.After the entry in 20～40 minutes four close shellfishes begin to discharge sperm in succession, keep seminal fluid that 500 milliliters of each parents discharge for the first time for future use with beaker respectively, abandon remaining seminal fluid and the parent and the container of hastening parturition are changed water and flushing repeatedly, all wash parent and container after after this discharging seminal fluid at every turn.The time that four parents discharge sperm continues 40～60 minutes respectively, and after the entry 2 hours, equal goodbye situations of discharging sperm separately not; After this, changed a fresh seawater in per 30 minutes and washed the parent and container, in 2.5～9.0 hours after entry, the phenomenon of pulse type emission ovum appears respectively in above-mentioned four parents, but sperm quantity significantly reduces.Collect the ovum of each time discharging in batches, look sperm quantity decision and whether wash ovum immediately and maybe need to add seminal fluid, when sperm quantity too much, on average can be observed 10 or more sperm around each ovum, need dilute or wash ovum as early as possible; Very few when sperm quantity, visible sperm only 1～2 the time, need be added the dilution seminal fluid of same individual around promptly average each ovum.H wherein
2Parent 2 times ovulation, the product of discharging is because of number of sperm deficiency in the water body, so add H one time for the first time
2The seminal fluid of parent discharging: the seminal fluid of getting prior preservation joins in the fertilization water body with getting 10ml after 10 times of the fresh seawater dilutions, and the ovum that merges twice discharging is hatched; H
23Ovulate altogether four times, sperm is excessive in the property product of the 4th discharge, abandons this batch ovum, merges first three batch ovum and hatches; H
24Three batches of equal sperms of ovum of discharging are excessive, are diluted to 150 liters respectively, wash then to merge behind the ovum to keep preceding two batches of ovums and hatch, and abandon the 3rd batch.H
10Ovulate altogether three times, the outer sperm quantity of visible average each egg mother cell of microscopically is respectively 15～22, washes the ovum that merges three batches behind the ovum respectively and hatches.
The fertilized egg that merges same parent is hatched and cultivation, and hatching is identical with embodiment 1 with breeding condition, and the female and male gametophyte of four parent's dischargings is all finished self-fertilization smoothly, removed H as a result
10Outer its excess-three parent has obtained the juvenile mollusk of the high 600 μ m of average shell.
H
1050,000 of the ovum sum deficiencies of discharging, three batches of sperm quantities around each ovum are respectively 22,15,20, the sperm around the ovum significantly reduces when directly hastening parturition than not injecting serotonin.H
10Because of the ovum number quantity not sufficient, obtain D shape larva amount seldom, can't continue to cultivate and abandon.
Table 2 Patinopecten yessoensis injection serotonin and gamete emission behaviour
| Family | Spermary area (%) | Serotonin concentration (μ mol/L) | Injection consumption (μ l) | The smart time of row (minute) | Ovulation period (hour) | Ovulation (criticizing) | Every batch of ovum number of sperm | |||
| 1 | 2 | 3 | 4 | |||||||
| H 2 H 23 H 24 H 10 | 15 20 30 50 | 50 20 50 500 | 200 200 100 100 | 40 35 20 30 | 4.5 3.0 2.5 9.0 | 2 4 3 3 | 2 8 12 22 | 4 6 14 15 | 9 18 20 | 14 |
Annotate: row's essence and ovulation period are the efflux time first of sperm and ovum; Number of sperm refers to around each egg mother cell of microscopically one the fixedly number of sperm on the focal plane.
Most marine shellfishes is capable in vitro fertilization.When in the prior art, in vitro fertilization in the water body sperm quantity cross the major general and influence fertilization rate; Otherwise the sperm quantity around the time of fertilization ovum is crossed at most and will be had a strong impact on hatching, can significantly increase the ratio of lopsided embryo and larva, even causes egg membrane molten broken.Suitable proportion between two kinds of gametes of the time of fertilization male and female is different between different shellfishes, but generally observing average one with microscopically fixedly has 3～6 sperms to be advisable around the egg mother cell on the focal plane, the ovum of haliotis discus hannai Ino is to the sperm quantity relative insensitivity, the sperm that tolerable is more sees with an ovum that 5～8 sperms are comparatively suitable, 10～15 sperms also are unlikely to influence and hatch outer Monday on the focal plane.In the embodiment of the invention 1, when not taking to inject serotonin, from H
2Number of sperm around its average every batch of ovum of five batches of emission product that the parent collects reaches 12～35 respectively, and after present embodiment takes to inject serotonin, and on average the outer number of sperm of each ovum only is 2 and 4 (table 2).The present embodiment explanation adopts the method for injection serotonin to impel sperm to excrete effectively in advance, the sperm quantity that discharges synchronously with ovum is significantly reduced, after adopting the technology of injection serotonin simultaneously, the milky zone account for the gonad gross area greater than this individual reproduction gland surface area summation 15%, close Becquerel smaller or equal to 30% to be to obtain the self progeny.And the milky zone reaches 50% H
10Do not obtain juvenile mollusk and be because the ovum quantity of its discharge is very few, if so the parent ovulation amount of arrenotoky gland ratio in 30～50% scopes can reach more than 300,000, then the method by the injection serotonin also is hopeful to obtain the self progeny.Surface area for the arrenotoky gland is the individuality (present embodiment is 15%) of this individual reproduction gland surface area summation 10～15%, is good with the method effect that adopts the injection serotonin then.
With flow cytometer, electron microscope and paraffin section technology to the parent H in embodiment 1 and 2
2, H
10The different parts of gonad tissue analyze respectively.All analysis results all prove, Chinese red zone wherein is that female reproduction glandular tissue, milky zone are the arrenotoky glandular tissue, structure is identical with normal dioecious gonad female or male Patinopecten yessoensis respectively, position, boundary in milky and Chinese red zone can be observed two kinds of reproductive cell of male and female and exist simultaneously.
Wherein, three peaks such as monoploid, dliploid, tetraploid appear with milky zone in the gonad of flow cytometer detection hermaphroditism Patinopecten yessoensis, indicated mature sperm, secondary spermatocyte and first spermatocyte respectively, come to the same thing with stream inspection dioecious, the male Patinopecten yessoensis of gonad normal appearance; Two peaks such as dliploid and tetraploid appear in the stream in tangerine zone inspection result in the gonad of hermaphroditism Patinopecten yessoensis, indicated ripe egg mother cell and oogonium respectively, come to the same thing with the inspection of the stream of the female Patinopecten yessoensis of dioecious gonad normal appearance.Tissue to milky zone in the hermaphroditic Patinopecten yessoensis gonad carries out scanning electron microscopic observation, finds the sperm structure indifference in sperm structure and the male Patinopecten yessoensis gonad of dioecious gonad normal appearance.With the paraffin section technology zone of the Chinese red in the hermaphroditic Patinopecten yessoensis gonad, milky and two kinds of color intersections is analyzed, found that wherein Chinese red position and the gonad of normal female individuals organizes identical, milky position to organize intersection identical, two kinds of colors then can observe two kinds of reproductive cell of male and female simultaneously with the gonad of normal male.The above results shows, outward appearance has the Patinopecten yessoensis of normal female (salmon pink) and male (milky) two kinds of gonad colors of Patinopecten yessoensis simultaneously, it is hermaphroditic individuality, zone with this species normal female individual reproduction gland same color (Chinese red) in the gonad is position, ovary place, and with the zone of normal male individual reproduction gland same color (milky) be the position at spermary place, two kinds of reproductive cell coexistences of its male and female and normal, grow synchronously.Therefore can discern, confirm hermaphroditic Patinopecten yessoensis special entity by the gonad appearance color.
Embodiment 3
Patinopecten yessoensis H
30And H
31Be the very little Patinopecten yessoensis of arrenotoky gland region area, H
30Only at the tip of gonad ox horn shape a milky spot is arranged, arrenotoky gland area accounts for 0.1% of gonad surface area summation, H
31A rotund milky patch is then arranged at gonadal lateral margin position, account for 0.5% of gonad surface area summation.The method that routine is hastened parturition is substantially the same manner as Example 1, hastens parturition H after drying in the shade 30 minutes in the ultraviolet seawater of 10 liters of preheatings
30And H
31Begin to discharge gamete respectively in entry 30 with after 55 minutes.Entry will be planted shellfish and be taken out after 1.5 hours, carefully stir water body and make the ovum suspension and sufficient chance and sperm combination are arranged, and the sampling microscopy is found H after 10 minutes
30Only can see sperm, H around 50% the ovum
31Around then being arranged, 85% ovum can see sperm.
With scalpel blade carefully with H
30And H
31Scratch respectively at gonadal white dot position, with carefully irrigate position and the sperm of going out being received in respectively in 200 milliliters the beaker of seawater, repeats above-mentioned steps repeatedly and obtained the as seen slightly milky seminal fluid of naked eyes.With H
30Seminal fluid divide all to join for four times and contain parent H
30In the property product of normal discharging, be separated by between per twice 3 minutes, add the seminal fluid front and back and all need stir the fertilization water body in order to avoid sperm skewness in water body.H
31Seminal fluid divide after then diluting 5 times and get 20 milliliters three times at every turn and join H
31In the property product of normal discharging, each 5 minutes at interval.At four cell stages statistics H
30And H
31It is 96.7% and 98.3% that the fertilization rate of inbred line is respectively.
According to prior art, the ovum of shellfish will form fertilization membrane finishing after fertilization, and is then insensitive to the sperm in the water body after forming fertilization membrane, so the measure that present embodiment is taked to increase seminal fluid in batches can be avoided the excessive problems such as embryo's deformity that cause of sperm.
Embodiment 4
The gonad feature and the Patinopecten yessoensis of chlamys farreri are very approaching, are the ox horn shape after promptly gonad is grown, and the female reproduction gland is Chinese red, malely then is milky, are easy to distinguish its sex in appearance from gonadal.The hermaphroditism of chlamys farreri is very rare simultaneously.
In the chlamys farreri colony of an artificial ripening, find the individuality of a gonad color exception certain year May, its gonad overall color is a Chinese red, scatter white point fragmentarily in the whole gonad, the diameter of point is about 0.1mm, and the estimation white portion accounts for 0.2% of the gonad gross area.This parent after drying in the shade 20 minutes, is hastened parturition with 23 ℃ ultraviolet irradiation seawater, entry 30 and change ultraviolet seawater after 50 minutes respectively 1 time, after began to discharge gamete in 10 minutes after once changing water.The property product that this kind shellfish is produced is divided into two groups of A, B, and wherein the A group is carried out self-fertilization, does not add the external source seminal fluid, and microscopically only 60% ovum is seen sperm on every side, and sperm quantity is seriously on the low side; The B group then adds the seminal fluid of a dioecious male chlamys farreri by the discharging of normally hastening parturition, and makes average each ovum sperm on every side reach 4.Four cell stage microscopies know that the fertilization rate of A group is 82.1% as a result, and the B group then reaches 98.6%.The chlamys farreri that contains Chinese red and two kinds of colors of milky in the The above results explanation gonad simultaneously is hermaphroditic special entity, and sperm that the hermaphroditism individuality of chlamys farreri is produced and ovum can self-fertilizations, and its female and male gametophyte is all normal on function.
Embodiment 5
The gonad of haliotis discus hannai Ino is the ox horn shape after growth, and the male and female individuality can be easy to distinguish by gonadal color under the normal condition, and wherein the gonad of female individuals is blackish green, and male then is milky.
Reach through artificial culture find altogether in sexually matured 2300 haliotis discus hannai Ino kind Baos that a gonad physically well develops, form is normal but the inhomogenous individuality of gonad surface color, this individuality is numbered No. 231.The gonad overwhelming majority zone of No. 231 haliotis discus hannai Inos is blackish green, but occurs the milky patch of small size in the gonad at position, right side, Bao body rear end, and this milky massive texture is nearly rectangle, and surface area accounts for 5% of gonad surface area summation.This kind Bao is picked up from the sea area, Miyagi Prefecture, Japan, cultivate through artificial ripening and hasten parturition when accumulated temperature reaches 1400 ° of D, the time body long 10.8cm, gonad plentiful.The accelerating cultivation water temperature of planting shellfish is 20 ℃.
Production method is carried out artificial induced spawning to this parent routinely: after drying in the shade 40 minutes, No. 231 kind Baos are placed the abalone groove of laying eggs separately, add 20 liters and be warming up in advance in 23 ℃ and the seawater, change 23 ℃ of seawater of a ultraviolet irradiation after 40 minutes through ultraviolet irradiation 600mW.h/L.Begin to discharge ovum and sperm after 50 minutes in kind of Bao entry, ovulation period continues 1.5 hours, discharges 30,000 in ovum altogether.Take out kind of a Bao, stirred a water body gently in per 10 minutes to make ovum be in floating state in after this 1 hour, step is hatched and larvae cultivation routinely then.
Obtain crawl 400 of larvas of later stage altogether, examine under a microscope normal, the healthy active larva of taking-up 42 forms wherein and collect seedling.Collect seedling and in 10 liters abalone hatching cylinder, carry out, on the wall of hatching cylinder, adhere to benthic diatom in advance, with 42 later stages selecting the larva that crawls be transferred in this cylinder, be that collector is directly collected seedling with the wall of hatching cylinder.This cylinder places under 20 ℃ of room temperatures, and hydrostatic is cultivated, and every day, full dose was changed water 1 time.Obtain to survive three of young Baos after three months, it is stripped down to forward on the black corrugated plating in an independent net cage, culture in a conventional manner.These three individualities were cultivated to April next year, and shell is long to reach 1.6,1.7 and 2.1cm respectively.Illustrate that the gonad outward appearance has blackish green simultaneously and haliotis discus hannai Inos two kinds of colors of milky are hermaphroditic individualities, its female and male gametophyte can be discharged synchronously by artificial induced spawning, and hermaphroditic Bao can be cultivated the self progeny by self-fertilization.
A kind ofly set up and cultivate the approach of inbred line then according to provided by the present invention by gonadal outward appearance being observed the hermaphroditism individuality of finding in the gonochorism type shellfish, can also implement to obtain the inbred line method for building up of the gonochorism type shellfish of ovum by post-mortem method, that is: oyster is a gonochorism type shellfish, it is higher that hermaphroditic frequency takes place, but the gonad of its male and female individuality is milky, therefore can not find hermaphroditic individuality to the observation of gonad color by naked eyes.Can normal development but oyster can obtain great-hearted female, male both sexes gamete and its by the approach of dissecting in conjunction with the fertilized egg that forms, therefore check the gonad tissue of each oyster one by one by microscope, be about to this individuality and dissect separately and obtain its gamete and make female, andro gamete fertilization in case find to have simultaneously the individuality of ovum and sperm, cultivate inbred line from same parent.This thinking is applicable to any shellfish that can obtain ovum by dissection, such as oyster, pearl shell etc.
Claims (5)
1. the method for building up of a gonochorism type shellfish inbred line is characterized in that: from gonochorism type shellfish, select have simultaneously two kinds of reproductive cell of male and female, be hermaphroditic special entity, adopt the strategy of selfing to set up and cultivate inbred line;
Described gonochorism type shellfish, its gonadal color same species female and male between have the difference that can under the live body situation, differentiate its sex by perusal;
Special entity in the described gonochorism type shellfish that has two kinds of reproductive cell of male and female simultaneously after its gonad is grown, can be recognized the gonad that this individuality has female, male two kinds of colors simultaneously by naked eyes in appearance from gonadal; And its gonad must be specially based on female: in surface area, the ratio that the arrenotoky gland accounts for the gonad gross area less than, equal 30%;
The method of inbred line is set up and cultivated to the strategy of described employing selfing, be specially: each parent is independently hastened parturition under the condition of mutually isolating and female and male gametophyte that same parent is produced carries out self-fertilization, hatch, cultivate and culture by the routine techniques of these species then.
2. according to the method for building up of the described gonochorism type of claim 1 shellfish inbred line, it is characterized in that: described independently hasten parturition and self-fertilization is meant that each parent impels it to discharge female and male gamete with the method for artificial stimulation separately in a container, make the ovum and the sperm fertilization of same individuality then, hasten parturition and fertilization process in by two kinds of gonadal relative scale situations of parent's male and female, taking measures artificial increases or reduces sperm quantity so that the relative scale of two kinds of gametes of male and female in the water body is carried out manual intervention and adjusting.
3. according to the method for building up of the described gonochorism type of claim 2 shellfish inbred line, it is characterized in that: the method that described relative scale to two kinds of gametes of male and female in the water body is carried out manual intervention and adjusting is specially: for the surface area of arrenotoky gland less than, equal the parent of this individual reproduction gland surface area summation 0.5%, hasten parturition in routine and to finish the back topography is carried out at position, parent's arrenotoky gland place, will join this individuality then by the seminal fluid that anatomic method obtains and hasten parturition in the property product that normally discharges by routine; For the surface area of arrenotoky gland greater than this individual reproduction gland surface area summation 15%, individuality smaller or equal to 30%, behind parent's dry in the shade stimulation, inject serotonin, in improve 3～5 ℃ ultraviolet irradiation seawater than former cultivation water temperature, hasten parturition then.
4. according to the method for building up of the described gonochorism type of claim 2 shellfish inbred line, it is characterized in that: the method that described relative scale to two kinds of gametes of male and female in the water body is carried out manual intervention and adjusting is specially: the surface area for the arrenotoky gland is the individuality of this individual reproduction gland surface area summation 10～15%, then adopts the method for injection serotonin.
5. according to the method for building up of claim 3 or 4 described gonochorism type shellfish inbred lines, it is characterized in that the consumption of described injection serotonin is: it is the serotonin solution of 20～500 μ mol/L that each parent injects 100～200 μ l concentration.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200410050568.0A CN1263374C (en) | 2004-10-10 | 2004-10-10 | Method for setting up dioecism type mussel inbred line |
| PCT/CN2005/001526 WO2006037263A1 (en) | 2004-10-10 | 2005-09-21 | Method for establishing gonochoristic shellfish selfing line |
| KR1020077010442A KR100868894B1 (en) | 2004-10-10 | 2005-09-21 | Method for establishing gonochoristic shellfish selfing line |
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| Application Number | Priority Date | Filing Date | Title |
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| CN200410050568.0A CN1263374C (en) | 2004-10-10 | 2004-10-10 | Method for setting up dioecism type mussel inbred line |
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| CN1628505A CN1628505A (en) | 2005-06-22 |
| CN1263374C true CN1263374C (en) | 2006-07-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN200410050568.0A Expired - Fee Related CN1263374C (en) | 2004-10-10 | 2004-10-10 | Method for setting up dioecism type mussel inbred line |
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| Country | Link |
|---|---|
| KR (1) | KR100868894B1 (en) |
| CN (1) | CN1263374C (en) |
| WO (1) | WO2006037263A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102318571B (en) * | 2011-08-05 | 2013-07-03 | 青岛农业大学 | Culture method of hybrid new variety of Argopecten irradias and Argopecten purpuratus |
| CN102440207B (en) * | 2011-10-10 | 2013-10-23 | 中国科学院南海海洋研究所 | Establishing method of Cnobilis inbred line |
| CN103478056B (en) * | 2013-10-23 | 2015-05-13 | 广东海洋大学 | Method for improving emission ratio of pinctada maxima parent gamete |
| CN103814845A (en) * | 2014-02-28 | 2014-05-28 | 中国科学院南海海洋研究所 | New method for building inbred lines of dioecious bivalve molluscs on large scale |
| CN111139237A (en) * | 2020-03-04 | 2020-05-12 | 大连海洋大学 | Extraction method and kit of Ruditapes philippinarum DNA |
| CN115053841A (en) * | 2022-07-12 | 2022-09-16 | 广东海洋大学 | Maturation promoting method for Xishi tongue parent shellfish |
| CN114885870B (en) * | 2022-07-12 | 2022-11-29 | 海南大学 | Spawning induction method for sipunculus australis |
| CN117898229A (en) * | 2024-03-19 | 2024-04-19 | 三亚热带水产研究院 | Artificial fertilization method for Chlamys nobilis |
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| CN1130970C (en) * | 2001-01-16 | 2003-12-17 | 中国科学院海洋研究所 | Method for preparing pure shellfish line |
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- 2005-09-21 WO PCT/CN2005/001526 patent/WO2006037263A1/en active Application Filing
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| CN1628505A (en) | 2005-06-22 |
| KR100868894B1 (en) | 2008-11-14 |
| KR20070067192A (en) | 2007-06-27 |
| WO2006037263A1 (en) | 2006-04-13 |
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