CN103931531A - Method for long-term storage of crassostrea hongkongensis eyespot larvas by critical low temperatures before freezing point - Google Patents
Method for long-term storage of crassostrea hongkongensis eyespot larvas by critical low temperatures before freezing point Download PDFInfo
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- CN103931531A CN103931531A CN201410153382.1A CN201410153382A CN103931531A CN 103931531 A CN103931531 A CN 103931531A CN 201410153382 A CN201410153382 A CN 201410153382A CN 103931531 A CN103931531 A CN 103931531A
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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
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- 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 dislcoses a method for long-term storage of crassostrea hongkongensis eyespot larvas by critical low temperatures before the freezing point. The method comprises the steps that a, the larvas are stored in a refrigeration mode, the concentrated crassostrea hongkongensis eyespot larvas are collected and are packaged and bundled into packages by bolting-silk meshes which is wetted by seawater, and then the packages are wrapped with sponges wetted by seawater and are placed at the temperature of 0-1 DEG C for refrigeration; b, according to the refrigerated eyespot larvas in the step a, seawater is sprayed on the sponges, and the eyespot larvas are made to keep wet; c, the larvas are unfrozen, the refrigerated eyespot larvas in the step b are taken out from the low temperature environments and are wetted by normal-temperature seawater, then the larvas are put in a container and evenly stirred, and the eyespot larvas can be obtained after the larvas are completely restored to be at the normal temperature. The eyespot larvas stored in the method almost do not suffer from frostbite, the seedling collecting efficiency can be effectively improved, and 90% and more of the eyespot larvas can achieve metamorphosis. The method provides a theory and practice foundation for studying cryobiology of the crassostrea hongkongensis larvas, dry larvas transportation, large-scale seedling synchronous production and the like. The method for long-term storage of the crassostrea hongkongensis eyespot larvas by the critical low temperatures before the freezing point has the advantages that operation is simple and convenient, and the practicability is high.
Description
Technical field:
The invention belongs to shellfish seed breeding technical field in modern marine agricultural, be specifically related to a kind of method of utilizing the front critical low temperature of freezing point to preserve for a long time Hong Kong oyster eyebot larvae.
Background technology:
Cryobiology is the emerging subject growing up the 1950's, and this subject is obtaining a wide range of applications (Chang Wancun etc., 1998) aspect the gamete of people, animal etc. and embryo's preservation at present.Although ultra-low temperature cold freeze techniques is widely applied in marine organisms, but because some halobiontic ovum is larger, and contain a large amount of yolk, oil droplet etc., increase the difficulty of freezing preservation, therefore the research of this respect concentrates on the low temperature of marine organisms sperm and preserves research upper (Li Yongren etc., 2005).
At present, cryopreserving sperm technology has obtained success on diversified economy fish, shrimp, shellfish, and obtains practical application.In shellfish gamete, embryo's preservation, also obtain breakthrough progress, particularly for the ultralow temperature Techniques of preserving of the early stage trochophore of oyster, reached business-like scale (Zhang Yan etc., 2004).Although, have indivedual report oyster larva low temperature short-term preservations researchs (Yu Ruihai etc., 2006), the relevant report that there is not yet low temperature and preserve for a long time Hong Kong oyster eyebot larvae.
Summary of the invention:
Be directed to the present situation that current Hong Kong oyster eyebot larvae there is no effectively long-term preservation measure, in conjunction with the low temperature resistant biological property of Hong Kong oyster eyebot larvae, the present invention by larva gather, the technological means such as larva refrigeration, larva management provides a kind of method that critical low temperature is preserved Hong Kong oyster eyebot larvae for a long time before freezing point of utilizing.
The method of utilizing the front critical low temperature of freezing point to preserve for a long time Hong Kong oyster eyebot larvae of the present invention, is characterized in that, comprises the following steps:
A, larva refrigeration: gather concentrated Hong Kong oyster eyebot larvae, it is installed and be bundled into bag with the wetting bolting silk net of seawater, then seal with the wetting sponge wrapping of seawater, be placed in refrigeration at 0-1 DEG C;
B, the eyebot larvae that step a is refrigerated spray seawater on its sponge, make its maintenance moistening; Generally can reach≤90d of cold preservation time.
C, larva are thawed: the eyebot larvae of step b refrigeration is taken out from low temperature environment, utilize normal temperature seawater wetting, put into afterwards container, stirred evenly, approximately, after 15-20min, until recovering completely after normal temperature, obtain eyebot larvae.In larva course of defrosting, 90% above eyebot larvae can swim in seawater, in existing state.The eyebot larvae of post-thaw survival can be dropped in the lower good adherance cement pit that fills fresh seawater and collects seedling.
The concentrated Hong Kong of the collection of described step a oyster eyebot larvae is to utilize 80 mesh sieve tulles (mesh maximum diameter of hole 250 μ are concentrated Hong Kong oyster eyebot larvae m).
The wetting bolting silk net of seawater of described step a is that (mesh maximum diameter of hole 20 μ m) for 500 mesh sieve tulles.
In described step a, gather concentrated Hong Kong oyster eyebot larvae, shell is high at 320-360 μ m, and shell length is at 270-300 μ m.
In described step a, Hong Kong oyster eyebot larvae is installed and is bundled into bag with the wetting bolting silk net of seawater, be limited in≤1kg of every bag eyebot larvae dress, prevents from that a large amount of larvas from pushing larva self is caused to damage.
The eyebot larvae to step a refrigeration of described step b sprays seawater on its sponge, makes its maintenance moistening, is to dewater in eyebot larvae body in order to prevent, makes its drying regime under moistening atmosphere, and air humidity maintains more than 100%.
The sprinkling seawater of described step b, its frequency be 5-7 days once.
In the research of Shellfish Larva low temperature Techniques of preserving, Li Yifeng etc. (2012) disclose the documents 1 of " the impact research that Trachyostracous mussel early larvae low temperature is preserved ".Documents 1, compared with the present invention, has following difference, 1. conservation object difference: what documents was preserved is Trachyostracous mussel, and the present invention's preservation is Hong Kong oyster.2. preserve stage difference: the D shape larva that documents 1 is preserved, early stage veliger; And the present invention's preservation is eyebot larvae, it is face later stage larva.3. preserving type difference: the low-density in seawater adopting when documents is preserved is preserved, preserves larva quantity and limited (≤25 larva/mL seawater) thereof; And the present invention adopt dry method preserve, can preserve a large amount of larvas (>=10 ten thousand/g).4. preserve management process difference: in documents 1 preservation process, need the bait of throwing something and feeding, complex steps; And the present invention does not need any bait of throwing something and feeding.
Yu Ruihai etc. (2006) disclose " the survival rate research of different budding Pacific oysters under different dry dewiness state " documents 2.Documents 2, compared with the present invention, has following difference, 1. conservation object difference: what documents 2 was preserved is long oyster, and the present invention's preservation is Hong Kong oyster.Long oyster is mainly distributed in region, northern China temperate zone, likes high salt low temperature full sea water environment, and annual production is 800,000 tons of left and right; And Hong Kong oyster is mainly distributed in the South China coastal subtropics and tropical area, the brackish water environment of hobby less salt high temperature, annual production is 1,300,000 tons of left and right.2. preserving type difference: the low temperature that documents 2 is used is 8-10 DEG C, and that the present invention's use is 0-1 DEG C.The 8-10 DEG C of middle low temperature environment for freezing in front low temperature, compares safer, but cannot reach the object of long-term preservation; And 0-1 DEG C be critical ultralow temperature before freezing point, those skilled in the art considered that under 0-1 DEG C of environment, larva may be freezed to death, before but the inventor finds 0-1 DEG C of freezing point, critical ultralow temperature can make eyebot larvae in shock state, thereby can preserve for a long time.3. holding time difference: documents 2 is short-term preservation, has only preserved 24h; And the present invention is long-term preservation, the holding time is 2-3 month.4. larva state difference: the long oyster larva in documents 2, in low temperature metabolism state, is only to have reduced metabolic rate; And the present invention makes the hibernation-like state of its eyebot larvae in cold shock formula, almost no longer carry out metabolism, but still maintain larva in condition of living organism.5. preservation effect difference: the eyebot larvae that documents 2 is preserved, its survival rate less than 90% after 24h, and the present invention long-term preserve 3 months with interior survival rate all more than 90%.6. preserve purposes difference: documents 2 is for being mainly applicable to long-distance transport in the short time, and main purpose of the present invention is in order to study Hong Kong oyster cryobiology and the tolerance to critical low temperature before freezing point thereof, thereby improve Hong Kong oyster larval metamorphosis rate, carry out large-scale production Hong Kong oyster healthy seedling.
The present invention is in conjunction with the low temperature resistant biological property of oyster larva, adopt cold shock mode, by larva gather, the technological means such as larva refrigeration, larva management, larva are thawed, created a kind of method that critical low temperature is preserved Hong Kong oyster eyebot larvae for a long time before freezing point of utilizing.The present invention's deepfreeze technology of utilizing antifreeze etc. to carry out different from the past, adopts first under 0-1 DEG C of cryogenic conditions and carries out larva refrigeration, can preserve in a large number eyebot larvae (>=1,000 hundred million)., almost not yet there is frostbite phenomenon in the eyebot larvae that the present invention preserves, can effectively improve the efficiency of collecting seedling, and more than 90% can complete metamorphosis (≤90d).The present invention provides theory and practice basis for studying Hong Kong oyster larva cryobiology, the transport of dry method larva, scale seed synchronous production etc.The present invention has the advantages such as easy and simple to handle, practical.
Embodiment
Following examples are to further illustrate of the present invention, but are not construed as limiting the invention.
Embodiment 1
A, larva gather: on May 30th, 2010, in the Zhuanghe's seashells nursery of Dalian, (mesh maximum diameter of hole 250 μ are concentrated 60m m) to utilize 80 bolting silk nets
3hong Kong oyster eyebot larvae in cement pit, obtains 6,000 ten thousand eyebot larvaes altogether, and its shell is high at 320-360 μ m.
B, larva refrigeration: (mesh maximum diameter of hole 20 μ m) install and are bundled into bag 500 mesh sieve tulles after eyebot larvae in step a is wetting with seawater, every bag≤1kg, outside utilizes sponge wrapping one deck of the moistening mistake of seawater afterwards, put into the incubator of 2L volume and seal, be positioned over afterwards 0-1 DEG C of refrigerator and cooled and hide.
C, larva management: utilized miniaturised nebuliser to spray seawater on its outer field sponge to packaged eyebot larvae in step b every 7 days, make it always in moisture state.
D, larva are thawed: after refrigeration 60d, first it is taken out from refrigerator, utilize normal temperature seawater to soak eyebot larvae after opening, put into afterwards 20L red listing, utilize dipper to stir evenly, approximately, after 15-20min, recover, after normal temperature, eyebot larvae to be dropped into 60m completely
3in cement pit, find that larva is close to 100% survival, normally ingests, moves about.After 15d, obtained fifty-five million oyster juvenile mollusk, distortion ratio is more than 90%.
Embodiment 2
A, larva gather: on June 10th, 2011, in Shenzhen Xu Lian Marine Bio Co., Ltd. nursery, (mesh maximum diameter of hole 250 μ are concentrated 30m m) to utilize 80 bolting silk nets
3hong Kong oyster eyebot larvae in cement pit, obtains 1,500 ten thousand eyebot larvaes altogether, and its shell is high at 320-360 μ m.
B, larva refrigeration: (mesh maximum diameter of hole 20 μ m) install and are bundled into bag 500 mesh sieve tulles after eyebot larvae in step a is wetting with seawater, every bag≤1kg, outside utilizes sponge wrapping one deck of the moistening mistake of seawater afterwards, put into the incubator of 2L volume and seal, be positioned over afterwards 0-1 DEG C of refrigerator and cooled and hide.
C, larva management: utilized miniaturised nebuliser to spray seawater on its outer field sponge to packaged eyebot larvae in step b every 7 days, make it always in moisture state.
D, larva are thawed: after refrigeration 36d, first it is taken out from refrigerator, utilize normal temperature seawater to soak eyebot larvae after opening, put into afterwards 20L red listing, utilize dipper to stir evenly, approximately, after 15-20min, recover, after normal temperature, to be dropped into 30m completely
3in cement pit, find that larva is close to 100% survival, normally ingests, moves about.After 15d, obtained 1,400 ten thousand oyster juvenile mollusks, distortion ratio is more than 90%.
Embodiment 3
A, larva gather: on June 15th, 2012, in Chinese Academy of Science Nanhai Ocean Research Institute's Zhanjiang marine economic animal experiment nursery, (mesh maximum diameter of hole 250 μ are concentrated 20m m) to utilize 80 bolting silk nets
3hong Kong oyster eyebot larvae in cement pit, obtains 1,000 ten thousand eyebot larvaes altogether, and its shell is high at 320-360 μ m.
B, larva refrigeration: (mesh maximum diameter of hole 20 μ m) install and are bundled into bag 500 mesh sieve tulles after eyebot larvae in step a is wetting with seawater, every bag≤1kg, outside utilizes sponge wrapping one deck of the moistening mistake of seawater afterwards, put into the incubator of 2L volume and seal, be positioned over afterwards 0-1 DEG C of refrigerator and cooled and hide.
C, larva management: utilized miniaturised nebuliser to spray seawater on its outer field sponge to packaged eyebot larvae in step b every 7 days, make it always in moisture state.
D, larva are thawed: after refrigeration 90d, first it is taken out from refrigerator, utilize normal temperature seawater to soak eyebot larvae after opening, put into afterwards the red bucket of 10L, utilize dipper to stir evenly, approximately, after 15-20min, recover, after normal temperature, to be dropped into 20m completely
3in cement pit, find that larva is close to 100% survival, normally ingests, moves about.After 15d, obtained 9,100,000 oyster juvenile mollusks, distortion ratio is more than 90%.
Claims (5)
1. a method of utilizing the front critical low temperature of freezing point to preserve for a long time Hong Kong oyster eyebot larvae, is characterized in that, comprises the following steps:
A, larva refrigeration: gather concentrated Hong Kong oyster eyebot larvae, it is installed and be bundled into bag with the wetting bolting silk net of seawater, then seal with the wetting sponge wrapping of seawater, be placed in refrigeration at 0-1 DEG C;
B, the eyebot larvae that step a is refrigerated spray seawater on its sponge, make its maintenance moistening;
C, larva are thawed: the eyebot larvae of step b refrigeration is taken out from low temperature environment, utilize normal temperature seawater wetting, put into afterwards container, stirred evenly, until recovering completely after normal temperature, obtain eyebot larvae.
2. method according to claim 1, is characterized in that, the concentrated Hong Kong of collection oyster eyebot larvae of described step a is to utilize the concentrated Hong Kong of 80 mesh sieve tulles oyster eyebot larvae.
3. method according to claim 1, is characterized in that, the wetting bolting silk net of seawater of described step a is 500 mesh sieve tulles.
4. method according to claim 1, is characterized in that, in described step a, Hong Kong oyster eyebot larvae is installed and is bundled into bag with the wetting bolting silk net of seawater, every be limited in≤1kg of bag eyebot larvae dress.
5. method according to claim 1, is characterized in that, the sprinkling seawater of described step b, its frequency be 5-7 days once.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106614242A (en) * | 2016-12-29 | 2017-05-10 | 广西还珠海洋生物科技有限公司 | Preservation method of seed ostrea rivularis transportation |
| CN106666239A (en) * | 2016-12-29 | 2017-05-17 | 广西还珠海洋生物科技有限公司 | Preservative for transporting young oysters of ostrea rivularis gould |
| CN106665456A (en) * | 2016-12-29 | 2017-05-17 | 广西还珠海洋生物科技有限公司 | Preservation method for ostrea rivularis young oyster transportation |
| CN106689116A (en) * | 2016-12-29 | 2017-05-24 | 广西还珠海洋生物科技有限公司 | Preservative for transporting young ostrea rivularis |
| CN109329145A (en) * | 2018-11-28 | 2019-02-15 | 中国水产科学研究院东海水产研究所 | A kind of transportation resources of shellfish larvae |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106614242A (en) * | 2016-12-29 | 2017-05-10 | 广西还珠海洋生物科技有限公司 | Preservation method of seed ostrea rivularis transportation |
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| CN106665456A (en) * | 2016-12-29 | 2017-05-17 | 广西还珠海洋生物科技有限公司 | Preservation method for ostrea rivularis young oyster transportation |
| CN106689116A (en) * | 2016-12-29 | 2017-05-24 | 广西还珠海洋生物科技有限公司 | Preservative for transporting young ostrea rivularis |
| CN109329145A (en) * | 2018-11-28 | 2019-02-15 | 中国水产科学研究院东海水产研究所 | A kind of transportation resources of shellfish larvae |
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