CN104336004A - Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms - Google Patents
Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms Download PDFInfo
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- CN104336004A CN104336004A CN201310314722.XA CN201310314722A CN104336004A CN 104336004 A CN104336004 A CN 104336004A CN 201310314722 A CN201310314722 A CN 201310314722A CN 104336004 A CN104336004 A CN 104336004A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention belongs to the technical field of marine organisms, and concretely relates to a method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms. The method comprises the following steps: carrying out illumination stimulation on male pacific oysters with the full growth sexual glands, dissecting the sexual glands, and filtering by a bolting silk net to obtain a large number of high-quality pacific oyster sperms; mixing a seminal fluid with an antifreeze liquid according to an appropriate ratio, packaging the obtained mixture in 2 ml or 5 ml cryopreserved tubes; carrying out balance incubation and programmed cooling, and preserving the obtained substances in liquid nitrogen for a long term; and carrying out water bath unfreezing, and carrying out natural water activation to obtain sperms with the frozen sperm vitality of above 70% and the fertilization rate of above 95%. The method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms has great significance to artificial breeding, germplasm preservation, genetic diversity and sustainable farming.
Description
Technical field
The invention belongs to field of marine biotechnology, specifically a kind of collection of high-quality Pacific oyster sperm and the method for freezen protective.
Background technology
Pacific oyster (Crassostrea gigas) is under the jurisdiction of Ostreidae, and huge oyster belongs to, and is commonly called as true oyster, is individual larger a kind of shellfish in oyster class.Its fine and tender taste, delicious flavour, nutritious, containing protein 45% ~ 57%, lipase 37 % ~ 11%, glycogen 19% ~ 38%, the content of iodine, higher than milk and egg, in addition, also containing multivitamin and trace element, has the laudatory title of ocean milk.The eighties in 20th century introduces China from Japan, Australia and Taiwan.The Excised Embryos of shellfish seminal fluid has great importance in aquaculture, genetic breeding and Germ-plasma resources protection.The artificial insemination of Pacific oyster many employings post-mortem method, requires higher to the male oyster of high-quality, and the cultivation that the quality of male close shellfish sperm seriously constrains Pacific oyster is bred.In addition, the Ultra-cryofreezing preservation of Pacific oyster sperm is the most important and reliable method that Pacific oyster is reserved seed for planting the building process male parent of family.Adopt the method for Ultra-cryofreezing preservation can in male Pacific oyster gonad development best period, by high-quality the sperm of close shellfish carry out frozen in batches and preserve for a long time, thaw at any time when needs and carry out artificial insemination.Excised Embryos can make the long-distance transportation of sperm be achieved simultaneously, is conducive to the hybridization of shellfish, seed selection, the acquisition of excellent shape and the protection of gene diversity.Therefore, the reliable method setting up Pacific oyster sperm Large Copacity Excised Embryos to the development of mariculture industry and the protection of bio-diversity significant.
Summary of the invention
The object of the present invention is to provide a kind of collection of high-quality Pacific oyster sperm and the method for Ultra-cryofreezing preservation.
For achieving the above object, the technical scheme that the present invention takes is:
A kind of collection of high-quality Pacific oyster sperm:
A) gonadal maturation, full Pacific oyster are carried out light stimulation, make male parent start ejaculation, collect sperm, stand-by;
B) by the sperm of above-mentioned collection through bolting silk net filtration, collect vigor higher than 80% sperm.
Described step a) selects shell long 8-10cm gonadal maturation, full Pacific oyster is supported temporarily, then pull the 1-1.5 hour that to be exposed to the sun under being placed in sunlight out, be turned to after irradiation after another side is exposed to the sun 1-1.5 hour again and put into 18-20 DEG C of nature seawater, start to be taken out from water immediately after ejaculation until male oyster.
Described step b) will gather sperm 100-120 mesh sieve tulle filter, collect vigor higher than 80% sperm.
The method that high-quality Pacific oyster sperm super-low temperature freezing is preserved,
A) vigor through silk cover filtering is added anti frozen liquid higher than in the sperm of 80%, hatch in 0-4 DEG C; Wherein seminal fluid mixes with the ratio of anti frozen liquid in 1:4-1:6 (V/V);
B) after sperm is hatched in anti frozen liquid, in separating device 2ml-5ml cryopreservation tube, in programmed cooling instrument through hatching, two step-down temperature, balance, then take out from programmed cooling instrument, directly put into the liquid nitrogen of-196 DEG C, preserve for a long time.
Described anti frozen liquid is made up of dilution, antifreeze, additive three part; The formula of dilution is: NaCl:8g/L, KCl:0.8g/L, Na
2hPO
42H
2o:0.1g/L, KH
2pO
4: 0.1g/L, NaHCO
3: 0.35g/L; Antifreeze: 10-12%(V/V) DMSO(dimethyl sulfoxide (DMSO)) and 3-4%PG(propane diols) (using dilution as the volume ratio of benchmark); Additive: glucose: 10g/L, trehalose 170g/L.
The vigor collected through silk cover filtering a) is added anti frozen liquid higher than in the sperm of 80% by described freezen protective step, firmly put upside down concussion centrifuge tube 5-10 time, leave standstill 2-3min, upper strata sperm after leaving standstill and the mixing material of anti frozen liquid are poured out, filtered by 100-120 mesh sieve tulle, filtrate is stand-by; The larger tissue block leached extrudes gently, residual sperm in tissue block is discharged, then anti frozen liquid washing screen tulle and remnant tissue 2-3 time is used, itself and above-mentioned filtrate are merged, finally supplement anti frozen liquid the ratio of sperm and anti frozen liquid to be arranged maintain 1:4-1:6, then hatch 20-30min in 0-4 DEG C.
Described freezen protective step b) sperm mixes with anti frozen liquid to be placed in programmed cooling instrument hatch 20-30min at 0-4 DEG C, then be cooled to-60--80 DEG C with the speed of-10--15 DEG C/min, and then be cooled to-150--180 DEG C with the speed of-20--25 DEG C/min, balance 2-5 minute, take out from programmed cooling instrument cavity, directly put into liquid nitrogen (-196 DEG C), packing is preserved.
It is cryopreservation tube is directly put into 30-40 DEG C of water-bath that the sperm of described freezen protective thaws, and is placed in room temperature, and concussion 20-30s, is then placed in 0-4 DEG C of refrigerator and preserves, stand-by.
The high-quality Pacific oyster of described freezen protective freezes essence when thawing, and directly put into 30-40 DEG C of water-bath 100-120s, period constantly shakes cryopreservation tube, and be then positioned over oscillator room temperature concussion 30s to melting completely, nature seawater activates.
Tool of the present invention has the following advantages:
1. the present invention gathers sperm is adopt stimulation of being exposed to the sun, and waits for the moment of oyster ejaculation, dissects sexual gland, can obtain the high-quality sperm of a large amount of high vigor;
2. filter with 100 mesh sieve tulles after the present invention gathers sperm, and the gonadal tissue of bulk farthest can be obtained the sperm in sexual gland by the extruding of bolting silk net;
3. the present invention adds anti frozen liquid in the process of semen collection, can reduce the open-assembly time of sperm at air, reduces the damage that sperm suffers before freezing, reduces the waste of sperm simultaneously, be conducive to the collection of sperm; And adopt DMSO and PG as antifreeze in anti frozen liquid, keep better permeability, and lower toxicity; Add 10g/L glucose in anti frozen liquid in addition, 170g/L trehalose, it can play a very good protection to the plasma membrane of sperm;
4. the present invention adopts 2-5ml cryopreservation tube volume comparatively large, and preservation sperm volume is large;
5. the present invention's freezing front fresh essence and anti frozen liquid when processing fully shakes mixing, and 0-4 DEG C balances 10-20min, make anti frozen liquid fully infiltrate sperm inside, the infringement to spermoblast when reducing cooling;
6. lower the temperature with segmented mode during freezing processing of the present invention, adopt and lower the temperature with-10---15 DEG C/min speed, frozen sperm is spent safely in " dangerous temperature district ", then the rate of temperature fall of-20 DEG C/min is adopted to enter stable state-150-180 DEG C fast, then drop into liquid nitrogen, cooling process is accurate and quick;
7. after frozen sperm of the present invention takes out from liquid nitrogen, adopt two step freezing processes, 30 DEG C-40 DEG C and 20 DEG C of two step are thawed, and sperm fast speed can be made to cross annealed zone, turn avoid the too high damage caused of local temperature simultaneously;
8. adopt the cooling process of precision of the present invention, repeated good stability, freeze essence thaw after anabiosis rate high, after activating, rate of motion is higher than 70%, and fertilization rate is substantially identical with fresh smart activity higher than 90%.
Embodiment
Embodiment 1
1.6 the middle of the month, on the occasion of Pacific oyster reproduction reproduction period, obtain coastal waters to raise in cages oyster 5 in cultivation base, Jiangnan, support temporarily in shellfish culture room, before freezen protective, male Pacific oyster is pulled out from storage pond, holding pond, be exposed to the sun under sunlight 1 hour, then be turned to after another side is exposed to the sun 1 hour again and put into room temperature seawater, take out immediately after observing male oyster ejaculation, take out seminal fluid by post-mortem method, silk cover filtering removes the assorted block of tissue, collect vigor higher than 80% sperm.
2. the collection vigor of above-mentioned collection is fully mixed higher than adding the anti frozen liquid prepared in advance in the sperm of 80%, firmly in trash ice, (0 DEG C) puts upside down concussion centrifuge tube 5-10 time gently, leave standstill 2min, upper strata sperm after leaving standstill and the mixing material of anti frozen liquid are poured out, by 100 bolting silk net filtrations, filtrate is stand-by; The larger tissue block leached extrudes gently, residual sperm in tissue block is discharged, then anti frozen liquid washing screen tulle and remnant tissue 2-3 time is used, itself and above-mentioned filtrate are merged, finally supplementary anti frozen liquid makes the ratio of sperm and anti frozen liquid arrange and maintains 1:5, then be mixed in 2ml cryopreservation tube, packing 12 cryopreservation tubes, 0-4 DEG C of balance 20min.Wherein, anti frozen liquid is made up of dilution, antifreeze, additive three part; The formula of dilution is: NaCl:8g/L, KCl:0.8g/L, Na
2hPO
42H
2o:0.1g/L, KH
2pO
4: 0.1g/L, NaHCO
3: 0.35g/L; Antifreeze is that 10%(is using dilution as the volume ratio v/v of benchmark) DMSO(dimethyl sulfoxide (DMSO)) and 3%PG(propane diols); Additive: glucose: 10g/L, trehalose 170g/L;
The layoutprocedure of anti frozen liquid: first configure dilution with distilled water, takes NaCl 8g, KCl 0.8g, Na
2hPO
42H
2o:0.1g/L, KH
2pO4:0.1g/L, NaHCO
3: 0.35g/L, is then settled to 1L for subsequent use; Liquid configuration antifreeze based on the dilution that employing has configured, wherein containing 10%DMSO(V:V) and 3%PG(V:V) (namely 100mL is containing 10mLDMSO, 3mL PG); Then by liquid based on the antifreeze that configured, add glucose 1g, trehalose 17g, is settled to 100mL.Anti frozen liquid is pre-configured and be placed in 4 DEG C of refrigerator precoolings;
3. programmed cooling instrument is opened; be chilled to 0 DEG C in advance; then sperm is mixed with anti frozen liquid to be placed in programmed cooling instrument and hatch 20min at 0 DEG C; then be cooled to-60 DEG C with the speed of-10 DEG C/min; and then be cooled to-150 DEG C with the speed of-20 DEG C/min; balance 2 minutes, poured into by all cryopreservation tubes and fill in the foam box of liquid nitrogen, then order is put into freezing storing box and is then put into the medium-term and long-term storage of liquid nitrogen container (-196 DEG C) one by one;
4. thaw after freezen protective 2h; choose arbitrarily 3 cryopreservation tubes to thaw; before thawing, freezing storing box is first placed in the incubation chamber filling liquid nitrogen; then cryopreservation tube is taken out fast from box and put into 37 DEG C of water-baths; shake 100s gently, be then placed in room temperature 20 DEG C and be positioned over oscillator concussion 30s to melting completely;
5. nature seawater activates, and microscopic examination thawn motility is higher than 70%; Carry out artificial insemination, obtain fertilization and reach more than 95%.
Embodiment 2
At the beginning of 1.7 months, on the occasion of Pacific oyster reproduction reproduction period, male Pacific oyster 1 is gathered in cultivation base, Jiangnan, gonad development is very full, in solar exposure 1 hour, then be turned to after another side is exposed to the sun 1 hour again and put into room temperature seawater, take out immediately after observing male oyster ejaculation, take out seminal fluid by post-mortem method; Silk cover filtering removes the assorted block of tissue, collect vigor higher than 80% sperm.
2. the collection vigor of above-mentioned collection is fully mixed higher than adding the anti frozen liquid prepared in advance in the sperm of 80%, firmly in trash ice, (0 DEG C) puts upside down concussion centrifuge tube 5-10 time gently, leave standstill 2min, upper strata sperm after leaving standstill and the mixing material of anti frozen liquid are poured out, by 100 bolting silk net filtrations, filtrate is stand-by; The larger tissue block leached extrudes gently; residual sperm in tissue block is discharged; then anti frozen liquid washing screen tulle and remnant tissue 2-3 time is used; itself and above-mentioned filtrate are merged; finally supplementary anti frozen liquid makes the ratio of sperm and anti frozen liquid arrange and maintains 1:4; then be mixed in 2ml cryopreservation tube, packing 25 cryopreservation tubes, 0-4 DEG C of balance 20min; Wherein, anti frozen liquid is made up of dilution, antifreeze, additive three part; The formula of dilution is: NaCl:8g/L, KCl:0.8g/L, Na
2hPO
42H
2o:0.1g/L, KH
2pO
4: 0.1g/L, NaHCO
3: 0.35g/L; Antifreeze is 10%DMSO(dimethyl sulfoxide (DMSO)) and 3%PG(propane diols); Additive: glucose: 10g/L, trehalose 170g/L;
The layoutprocedure of anti frozen liquid: first configure dilution with distilled water, takes NaCl 8g, KCl 0.8g, Na
2hPO
42H
2o:0.1g/L, KH
2pO4:0.1g/L, NaHCO
3: 0.35g/L, is then settled to 1L for subsequent use; Liquid configuration antifreeze based on the dilution that employing has configured, wherein containing 10%DMSO(V:V) and 3%PG(V:V) (namely 100mL is containing 10mLDMSO, 3mL PG); Then by liquid based on the antifreeze that configured, add glucose 1g, trehalose 17g, is settled to 100mL.Anti frozen liquid is pre-configured and be placed in 4 DEG C of refrigerator precoolings;
3. programmed cooling instrument is opened; be chilled to 0 DEG C in advance; then sperm is mixed with anti frozen liquid to be placed in programmed cooling instrument and hatch 20min at 0 DEG C; then be cooled to-80 DEG C with the speed of-15 DEG C/min; and then be cooled to-150 DEG C with the speed of-20 DEG C/min; balance 5 minutes, poured into by all cryopreservation tubes and fill in the foam box of liquid nitrogen, then order is put into freezing storing box and is then put into the medium-term and long-term storage of liquid nitrogen container (-196 DEG C) one by one;
4. thaw after freezen protective 2h, choose arbitrarily 3 cryopreservation tubes to thaw, before thawing, freezing storing box is first placed in the incubation chamber filling liquid nitrogen, then cryopreservation tube is taken out fast from box and put into 37 DEG C of water-baths, shake 100s gently, be then placed in room temperature 20 DEG C and be positioned over oscillator concussion 30s to melting completely;
5. nature seawater activates, and microscopic examination thawn motility is higher than 70%; Artificial insemination, obtains fertilization and reaches more than 95%;
6. remaining sample is whole to the medium-term and long-term preservation of liquid nitrogen container, and is used successfully to the structure of Pacific oyster family.
Claims (9)
1. a collection for high-quality Pacific oyster sperm, is characterized in that:
A) gonadal maturation, full Pacific oyster are carried out light stimulation, make male parent start ejaculation, collect sperm, stand-by;
B) by the sperm of above-mentioned collection through bolting silk net filtration, collect vigor higher than 80% sperm.
2. by the collection of high-quality Pacific oyster sperm according to claim 1, it is characterized in that: described step a) selects shell long 8-10cm gonadal maturation, full Pacific oyster is supported temporarily, then pull the 1-1.5 hour that to be exposed to the sun under being placed in sunlight out, be turned to after irradiation after another side is exposed to the sun 1-1.5 hour again and put into 18-20 DEG C of nature seawater, start to be taken out from water immediately after ejaculation until male oyster.
3. by the collection of high-quality Pacific oyster sperm according to claim 1, it is characterized in that: the sperm 100-120 mesh sieve tulle that described step b) will gather filters, collect vigor higher than 80% sperm.
4. a method for high-quality Pacific oyster sperm super-low temperature freezing preservation, is characterized in that:
A) vigor through silk cover filtering is added anti frozen liquid higher than in the sperm of 80%, hatch in 0-4 DEG C; Wherein seminal fluid mixes with the ratio of anti frozen liquid in 1:4-1:6 (V/V);
B) after sperm is hatched in anti frozen liquid, in separating device 2ml-5ml cryopreservation tube, in programmed cooling instrument through hatching, two step-down temperature, balance, then take out from programmed cooling instrument, directly put into the liquid nitrogen of-196 DEG C, preserve for a long time.
5., by the method that high-quality Pacific oyster sperm super-low temperature freezing according to claim 4 is preserved, it is characterized in that: described anti frozen liquid is made up of dilution, antifreeze, additive three part; The formula of dilution is: NaCl:8g/L, KCl:0.8g/L, Na
2hPO
42H
2o:0.1g/L, KH
2pO
4: 0.1g/L, NaHCO
3: 0.35g/L; Antifreeze: 10-12%(V/V) DMSO(dimethyl sulfoxide (DMSO)) and 3-4%PG(propane diols); Additive: glucose: 10g/L, trehalose 170g/L.
6., by the method that high-quality Pacific oyster sperm super-low temperature freezing according to claim 4 is preserved, it is characterized in that:
The vigor collected through silk cover filtering a) is added anti frozen liquid higher than in the sperm of 80% by described step, firmly put upside down concussion centrifuge tube 5-10 time, leave standstill 2-3min, the mixing material of the upper strata sperm after standing and anti frozen liquid is poured out, filtered by 100-120 mesh sieve tulle, filtrate is stand-by; The larger tissue block leached extrudes gently, residual sperm in tissue block is discharged, then anti frozen liquid washing screen tulle and remnant tissue 2-3 time is used, itself and above-mentioned filtrate are merged, finally supplement anti frozen liquid the ratio of sperm and anti frozen liquid to be arranged maintain 1:4-1:6, then hatch 20-30min in 0-4 DEG C.
7., by the method that high-quality Pacific oyster sperm super-low temperature freezing according to claim 4 is preserved, it is characterized in that:
Described step b) sperm mixes with anti frozen liquid to be placed in programmed cooling instrument hatch 20-30min at 0-4 DEG C, then be cooled to-60--80 DEG C with the speed of-10--15 DEG C/min, and then be cooled to-150--180 DEG C with the speed of-20--25 DEG C/min, balance 2-5 minute, take out from programmed cooling instrument cavity, directly put into liquid nitrogen (-196 DEG C), packing is preserved.
8., by the method that high-quality Pacific oyster sperm super-low temperature freezing according to claim 4 is preserved, it is characterized in that:
It is cryopreservation tube is directly put into 30-40 DEG C of water-bath that the sperm of described freezen protective thaws, and is placed in room temperature, and concussion 20-30s, is then placed in 0-4 DEG C of refrigerator and preserves, stand-by.
9., by the method that high-quality Pacific oyster sperm super-low temperature freezing according to claim 8 is preserved, it is characterized in that:
The high-quality Pacific oyster of described freezen protective freezes essence when thawing, and directly put into 30-40 DEG C of water-bath 100-120s, period constantly shakes cryopreservation tube, and be then positioned over oscillator room temperature concussion 30s to melting completely, nature seawater activates.
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| CN201310314722.XA CN104336004A (en) | 2013-07-24 | 2013-07-24 | Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110326610A (en) * | 2019-07-19 | 2019-10-15 | 大连海洋大学 | Sea cucumber sperm cryopreservation method |
| CN112075415A (en) * | 2020-09-25 | 2020-12-15 | 中国科学院海洋研究所 | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms |
| CN116076485A (en) * | 2022-12-26 | 2023-05-09 | 中国海洋大学 | Ultra-low temperature freezing protection liquid for grape dental oyster sperm and ultra-low temperature freezing preservation method |
| 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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2013
- 2013-07-24 CN CN201310314722.XA patent/CN104336004A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110326610A (en) * | 2019-07-19 | 2019-10-15 | 大连海洋大学 | Sea cucumber sperm cryopreservation method |
| CN110326610B (en) * | 2019-07-19 | 2021-09-24 | 大连海洋大学 | Ultralow temperature cryopreservation method for sea cucumber sperms |
| CN112075415A (en) * | 2020-09-25 | 2020-12-15 | 中国科学院海洋研究所 | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms |
| CN112075415B (en) * | 2020-09-25 | 2021-08-31 | 中国科学院海洋研究所 | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms |
| CN116076485A (en) * | 2022-12-26 | 2023-05-09 | 中国海洋大学 | Ultra-low temperature freezing protection liquid for grape dental oyster sperm and ultra-low temperature freezing preservation method |
| 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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Inventor after: Liu Qinghua Inventor after: Li Jun Inventor after: Xu Fei Inventor after: Zhang Guofan Inventor after: Li Li Inventor after: Han Longjiang Inventor before: Liu Jianguo Inventor before: Li Jun Inventor before: Xu Fei Inventor before: Zhang Guofan Inventor before: Li Li Inventor before: Han Longjiang |
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Free format text: CORRECT: INVENTOR; FROM: LIU JIANGUO LI JUN XU FEI ZHANG GUOFAN LI LI HAN LONGJIANG TO: LIU QINGHUA LI JUN XU FEI ZHANG GUOFAN LI LI HAN LONGJIANG |
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Application publication date: 20150211 |