CN103931531B - A kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time - Google Patents
A kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time Download PDFInfo
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- CN103931531B CN103931531B CN201410153382.1A CN201410153382A CN103931531B CN 103931531 B CN103931531 B CN 103931531B CN 201410153382 A CN201410153382 A CN 201410153382A CN 103931531 B CN103931531 B CN 103931531B
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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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Abstract
The invention discloses a kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time.It comprises a, larva refrigeration: gather concentrated Hong Kong oyster eyebot larvae, it is installed with the bolting silk net that seawater is wetting and is bundled into bag, then seal with the sponge wrapping that seawater is wetting, refrigerate at being placed in 0-1 DEG C; B, to step a refrigeration eyebot larvae spray seawater on its sponge, make it keep moistening; C, larva are thawed: taken out from low temperature environment by the eyebot larvae that step b refrigerates, and utilize normal temperature seawater to soak, put into container afterwards, stirred evenly, after recovering normal temperature completely, namely obtain eyebot larvae.The eyebot larvae that the present invention preserves, almost not yet occur frostbite phenomenon, effectively can improve efficiency of collecting seedling, more than 90% can complete metamorphosis.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.
Description
Technical field:
The invention belongs to shellfish seed breeding technical field in modern marine agricultural, be specifically related to a kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time.
Background technology:
Cryobiology is the emerging subject of of growing up the 1950's, and this subject obtains a wide range of applications (Chang Wancun etc., 1998) in the gamete of people, animal etc. and the preservation of embryo at present.Although ultra-low temperature cold freeze techniques is widely applied in marine organisms, but because some halobiontic ovum is larger, and containing a large amount of yolk, oil droplet etc., add the difficulty of freezen protective, therefore the research of this respect concentrates on the Cord blood research upper (Li Yongren etc., 2005) of marine organisms sperm.
At present, cryopreserving sperm technology obtains successfully on diversified economy fish, shrimp, shellfish, and obtains practical application.The preservation of shellfish gamete, embryo also achieves breakthrough progress, particularly for the Excised Embryos technology of the early stage trochophore of oyster, reaches business-like scale (Zhang Yan etc., 2004).Although, have indivedual report oyster larva low temperature short-term preservation research (Yu Ruihai etc., 2006), there is not yet the relevant report that low temperature preserves Hong Kong oyster eyebot larvae for a long time.
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 biological property that Hong Kong oyster eyebot larvae is low temperature resistant, the present invention by the technological means such as larva collection, larva refrigeration, larva management provide a kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time.
Of the present invention utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time, it is characterized in that, comprise the following steps:
A, larva refrigeration: gather concentrated Hong Kong oyster eyebot larvae, install it with the bolting silk net that seawater is wetting and be bundled into bag, then seal with the sponge wrapping that seawater is wetting, refrigerate at being placed in 0-1 DEG C;
B, to step a refrigeration eyebot larvae spray seawater on its sponge, make it keep moistening; Cold preservation time generally can reach≤90d.
C, larva are thawed: taken out from low temperature environment by the eyebot larvae that step b refrigerates, and utilize normal temperature seawater to soak, put into container afterwards, stirred evenly, after about 15-20min, after recovering normal temperature completely, namely obtain eyebot larvae.In larva course of defrosting, more than 90% eyebot larvae can swim in the seawater, is in existing state.The eyebot larvae of post-thaw survival can be dropped into fill in the lower good adherance cement pit of fresh seawater and collect seedling.
It is utilize 80 mesh sieve tulles (250 μm, mesh maximum diameter of hole) concentrated Hong Kong oyster eyebot larvae that the collection of described step a concentrates Hong Kong oyster eyebot larvae.
The bolting silk net that the seawater of described step a is wetting is 500 mesh sieve tulles (20 μm, mesh maximums diameter of hole).
Gather concentrated Hong Kong oyster eyebot larvae in described step a, shell is high at 320-360 μm, and shell length is at 270-300 μm.
Installed by the bolting silk net that Hong Kong oyster eyebot larvae seawater is wetting in described step a and be bundled into bag, often bag eyebot larvae dress is limited in≤1kg, prevents a large amount of larva from pushing and causes damage to larva self.
Described step b sprays seawater on its sponge to the eyebot larvae of step a refrigeration, and make it keep moistening, be dewater in eyebot larvae body to prevent, the drying regime under making it be in moistening atmosphere, air humidity maintains more than 100%.
The sprinkling seawater of described step b, its frequency be 5-7 days once.
In the technical research of Shellfish Larva Cord blood, the documents 1 that Li Yifeng etc. (2012) disclose " influence research of Trachyostracous mussel early larvae Cord blood ".Documents 1, compared with the present invention, has following difference, and 1. conservation object is different: what documents was preserved is Trachyostracous mussel, and the present invention's preservation is Hong Kong oyster.2. the stage of preserving is different: the D shape larva that documents 1 is preserved, namely early stage veliger; And the present invention's preservation is eyebot larvae, it is face later stage larva.3. preserving type is different: the low-density in the seawater adopted when documents is preserved is preserved, and preserves larva quantity and limited (≤25 larva/mL seawater) thereof; And the dry method that the present invention adopts is preserved, and can preserve a large amount of larva (>=10 ten thousand/g).4. management process is preserved different: in documents 1 preservation process, need bait of throwing something and feeding, complex steps; And the present invention does not need any bait of throwing something and feeding.
Documents 2 that Yu Ruihai etc. (2006) disclose " the survival rate research of Pacific oyster under different dry dewiness state of different growing periods ".Documents 2, compared with the present invention, has following difference, and 1. conservation object is different: what documents 2 was preserved is long oyster, and the present invention's preservation is Hong Kong oyster.Long oyster is mainly distributed in northern China temperate regions, and like high salt low temperature full sea water environment, annual production is at about 800,000 tons; 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 at about 1,300,000 tons.2. preserving type is different: the low temperature that documents 2 uses is 8-10 DEG C, and the present invention uses 0-1 DEG C.The 8-10 DEG C of middle low temperature environment for freezing in front low temperature, comparatively speaking safer, but long-term object of preserving cannot be reached; And 0-1 DEG C is critical ultra low temperature before freezing point, those skilled in the art considered that under 0-1 DEG C of environment, larva may be freezed to death, but before the present inventor finds 0-1 DEG C of freezing point, critical ultra low temperature can make eyebot larvae be in shock state, thus can preserve for a long time.3. the holding time is different: documents 2 is short-term preservation, only saves 24h; And the present invention is long-term preservation, the holding time is 2-3 month.4. larva state is different: the long oyster larva in documents 2 is in low temperature metabolism state, is only reduction of metabolic rate; And the present invention makes its eyebot larvae be in the hibernation-like state of cold shock formula, almost no longer carry out metabolism, but still maintenance larva is in condition of living organism.5. preservation effect is different: the eyebot larvae that documents 2 is preserved, and after 24h, its survival rate is less than 90%, and the present invention preserves survival rate within 3 months for a long time all more than 90%.6. purposes is preserved different: documents 2 is for being mainly applicable to long-distance transport in the short time, and main purpose of the present invention is to study Hong Kong oyster cryobiology and the tolerance to low critical temperature before freezing point thereof, thus 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, gathered by larva, larva refrigeration, larva management, larva such as to thaw at the technological means, create a kind of utilize freezing point before low critical temperature preserve the method for Hong Kong oyster eyebot larvae for a long time.The present invention's deepfreeze technology utilizing antifreeze etc. to carry out different from the past, carries out larva refrigeration under adopting 0-1 DEG C of cryogenic conditions first, can preserve eyebot larvae (>=1,000 hundred million) in a large number.The eyebot larvae that the present invention preserves, almost not yet occur frostbite phenomenon, effectively can improve efficiency of collecting seedling, 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 further illustrate of the present invention, but be not construed as limiting the invention.
Embodiment 1
A, larva gather: on May 30th, 2010, in the Zhuanghe's seashells nursery of Dalian, utilize 80 bolting silk nets (250 μm, mesh maximum diameter of hole) to concentrate 60m
3hong Kong oyster eyebot larvae in cement pit, obtain 6,000 ten thousand eyebot larvaes altogether, its shell is high at 320-360 μm.
B, larva refrigeration: the 500 mesh sieve tulles (20 μm, mesh maximum diameter of hole) after being soaked by eyebot larvae seawater in step a install and are bundled into bag, often bag≤1kg, outside utilizes sponge wrapping one deck of the moistening mistake of seawater afterwards, the incubator putting into 2L volume is sealed, and is positioned over 0-1 DEG C of refrigerator and cooled afterwards and hides.
C, larva management: utilized miniaturised nebuliser to spray seawater on its outer field sponge to eyebot larvae packaged in step b every 7 days, make it be in moisture state always.
D, larva are thawed: after refrigeration 60d, first it taken out from refrigerator, utilize normal temperature seawater to soak eyebot larvae after opening, put into 20L red listing afterwards, utilize dipper to stir evenly, and after about 15-20min, after recovering normal temperature completely, eyebot larvae are dropped into 60m
3in cement pit, find that larva is close to 100% survival, normally ingest, move about.After 15d, obtain 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, utilize 80 bolting silk nets (250 μm, mesh maximum diameter of hole) to concentrate 30m
3hong Kong oyster eyebot larvae in cement pit, obtain 1,500 ten thousand eyebot larvaes altogether, its shell is high at 320-360 μm.
B, larva refrigeration: the 500 mesh sieve tulles (20 μm, mesh maximum diameter of hole) after being soaked by eyebot larvae seawater in step a install and are bundled into bag, often bag≤1kg, outside utilizes sponge wrapping one deck of the moistening mistake of seawater afterwards, the incubator putting into 2L volume is sealed, and is positioned over 0-1 DEG C of refrigerator and cooled afterwards and hides.
C, larva management: utilized miniaturised nebuliser to spray seawater on its outer field sponge to eyebot larvae packaged in step b every 7 days, make it be in moisture state always.
D, larva are thawed: after refrigeration 36d, first it taken out from refrigerator, utilize normal temperature seawater to soak eyebot larvae after opening, put into 20L red listing afterwards, utilize dipper to stir evenly, and after about 15-20min, after recovering normal temperature completely, are dropped into 30m
3in cement pit, find that larva is close to 100% survival, normally ingest, move about.After 15d, obtain 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, utilize 80 bolting silk nets (250 μm, mesh maximum diameter of hole) to concentrate 20m
3hong Kong oyster eyebot larvae in cement pit, obtain 1,000 ten thousand eyebot larvaes altogether, its shell is high at 320-360 μm.
B, larva refrigeration: the 500 mesh sieve tulles (20 μm, mesh maximum diameter of hole) after being soaked by eyebot larvae seawater in step a install and are bundled into bag, often bag≤1kg, outside utilizes sponge wrapping one deck of the moistening mistake of seawater afterwards, the incubator putting into 2L volume is sealed, and is positioned over 0-1 DEG C of refrigerator and cooled afterwards and hides.
C, larva management: utilized miniaturised nebuliser to spray seawater on its outer field sponge to eyebot larvae packaged in step b every 7 days, make it be in moisture state always.
D, larva are thawed: after refrigeration 90d, first it taken out from refrigerator, utilize normal temperature seawater to soak eyebot larvae after opening, put into the red bucket of 10L afterwards, utilize dipper to stir evenly, and after about 15-20min, after recovering normal temperature completely, are dropped into 20m
3in cement pit, find that larva is close to 100% survival, normally ingest, move about.After 15d, obtain 9,100,000 oyster juvenile mollusks, distortion ratio is more than 90%.
Claims (5)
1. before utilizing freezing point, low critical temperature preserves a method for Hong Kong oyster eyebot larvae for a long time, it is characterized in that, comprises the following steps:
A, larva refrigeration: gather concentrated Hong Kong oyster eyebot larvae, install it with the bolting silk net that seawater is wetting and be bundled into bag, then seal with the sponge wrapping that seawater is wetting, refrigerate at being placed in 0-1 DEG C;
B, to step a refrigeration eyebot larvae spray seawater on its sponge, make it keep moistening;
C, larva are thawed: taken out from low temperature environment by the eyebot larvae that step b refrigerates, and utilize normal temperature seawater to soak, put into container afterwards, stirred evenly, after recovering normal temperature completely, namely obtain eyebot larvae.
2. method according to claim 1, is characterized in that, it is utilize 80 mesh sieve tulles to concentrate Hong Kong oyster eyebot larvae that the collection of described step a concentrates Hong Kong oyster eyebot larvae.
3. method according to claim 1, is characterized in that, the bolting silk net that the seawater of described step a is wetting is 500 mesh sieve tulles.
4. method according to claim 1, is characterized in that, is installed by the bolting silk net that Hong Kong oyster eyebot larvae seawater is wetting and be bundled into bag, the often weight≤1kg of bag eyebot larvae in described step a.
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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| 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 |
| 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 |
| 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 |
| 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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Patent Citations (3)
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| DE4205386C1 (en) * | 1992-02-19 | 1992-12-10 | Rainer Dipl.-Biol. 1000 Berlin De Bock | Cryo:preservation and revitalisation of fish eggs, crab larvae etc. - involves treating with e.g. DMSO, freezing and revitalising by warming |
| CN1071803A (en) * | 1992-12-22 | 1993-05-12 | 俞嘉麒 | The method of keeping fish or shrimp alive by hibernating and reviving |
| CN101946724A (en) * | 2010-08-10 | 2011-01-19 | 上海海洋大学 | Method for cold preservation of mytilus coruscus D-shape larvas |
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