CN103348966A - Method for efficient ultralow temperature cryopreservation of turbot sperms - Google Patents
Method for efficient ultralow temperature cryopreservation of turbot sperms Download PDFInfo
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- CN103348966A CN103348966A CN2013102130128A CN201310213012A CN103348966A CN 103348966 A CN103348966 A CN 103348966A CN 2013102130128 A CN2013102130128 A CN 2013102130128A CN 201310213012 A CN201310213012 A CN 201310213012A CN 103348966 A CN103348966 A CN 103348966A
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Abstract
The invention belongs to the technical field of marine organisms, and concretely relates to a method for the efficient ultralow temperature cryopreservation of turbot sperms. The method comprises the following steps: mixing the seminal fluid of turbots with an antifreeze liquid according to a volume ratio of 3:1-4:1, carrying out two-step cooling treatment, carrying out one-step balancing, adding to liquid nitrogen (having a temperature of -196DEG C), and preserving; and the antifreeze liquid is composed of a diluent, an antifreeze agent and an additive. High-quality frozen sperms obtained through the method for the efficient ultralow temperature cryopreservation of turbot sperms are of great significance of the artificial breeding, germplasm preservation, heredity diversity and sustainable culturing of the turbots.
Description
Technical field
The invention belongs to the marine biotechnology field, the method for the specifically freezing preservation of a kind of turbot sperm high-efficiency ultralow temperature.
Background technology
Turbot (Scophthalmus maximus) is under the jurisdiction of Bothidae, brill belongs to, and English name Turbot claims again " how precious fish ", the distinctive flatfish of a kind of Coast of Europe, belong to cold warm nature fish, it is littoral mainly to be distributed in northeast, the Atlantic Ocean, and also there is distribution in Black Sea, the Baltic Sea and Mediterranean.Because its fast growth, delicious meat, disease is less and be convenient to dense stocking, is the desirable kind of industrial aquaculture.China introduced the turbot seed from Britain first in 1992, support and artificial propagation by domestication, examination, broke through the yielding ability seedling growing process in 1999, the turbot aquaculture obtains flourish in China, set up at present a whole set of parent population regulation and control, artificial seed's cultivation and industrial aquaculture production technology.Turbot has become one of most important marine fish culture kind of northern China at present, has high commercial value.Because it is short that the turbot milter produces the smart time, produce the characteristics such as smart amount is few, seriously restricted the acquisition of its high quality fertilized egg of scophthatmus and the output of seed.Freezing turbot sperm can be regulated and control the male turbot parent population of a collection of maturation by artificial gonad development, concentrated batch is preserved high-quality turbot seminal fluid, thaw when needed and carry out artificial insemination acquisition fertilized egg, breeding the phase high-quality seminal fluid difficulty that supply falls short of demand thereby overcome turbot.In recent years, because the continuous expansion of cultivation scale, to the continuous expansion of turbot germplasm demand, the sperm freezing technology of turbot is badly in need of setting up in addition.It is all significant to the protection of the development that promotes mariculture industry and germ plasm resource to set up reliable method that the large capacity ultralow temperature of turbot sperm preserves.
Summary of the invention
The object of the present invention is to provide the method for the freezing preservation of a kind of turbot sperm high-efficiency ultralow temperature.
For achieving the above object, the technical scheme taked of the present invention is:
The method of the freezing preservation of a kind of turbot sperm high-efficiency ultralow temperature, with the seminal fluid of turbot and anti frozen liquid by volume the ratio of 3:1-4:1 mix, process through the cooling of two steps after the mixing, put into liquid nitrogen (196 ℃) after the step balance, packing is preserved; Anti frozen liquid is comprised of dilution, antifreeze, additive three parts.
Wherein, dilution is NaCl8g/L, KCl0.4g/L, MgSO
47H
2O0.1g/L, MgCl
26H
2O0.1g/L, Na
2HPO
42H
2O0.06g/L, KH
2PO
40.06g/L, NaHCO
30.35g/L; Antifreeze is final concentration 60%(V/V) the DMSO(dimethyl sulfoxide (DMSO)) or final concentration 60%(V/V), propane diols (PG); Additive: the yolk (V/V) of sucrose 34.2g/L and final concentration 10%.
Further, the layoutprocedure of anti frozen liquid: at first dispose dilution with distilled water, weigh NaCl8g, KCl0.4g, MgSO
47H
2O0.1g, MgCl
26H
2O0.1g, Na
2HPO
42H
2O0.06g, KH
2PO
40.06g, NaHCO
30.35g it is for subsequent use then to be settled to 1L; Adopt the dilution disposed to dispose anti frozen liquid as basal liquid, wherein contain 60%DMSO(V:V) (being that the 100mL anti frozen liquid contains 60mLDMSO) or 60%PG(V:V) (being that the 100mL anti frozen liquid contains 60ml PG); The anti frozen liquid that then will configure adds sucrose 3.42g as basal liquid, and yolk 10ml is settled to 100mL.
Described anti frozen liquid is positioned over before use in 0 ℃ of refrigerator precooling and spends the night, and is stand-by.The seminal fluid of described turbot through the anti frozen liquid mixed diluting places the cryopreservation tube of 2ml or 5ml.
The seminal fluid of described turbot and anti frozen liquid be (0 ℃) light shaking, abundant mixing 30s-1min in trash ice, then be cooled to-80 ℃ with-8 ℃/min, then be cooled to-120 ℃ with-10 ℃/min, balance is put into liquid nitrogen (196 ℃) after 2 minutes, and packing is preserved.
Packing is stored in turbot in the liquid nitrogen freezes the row that progresses greatly and thaw, concrete grammar is: the cryopreservation tube that will have a seminal fluid is directly put into the 37-40 ℃ of water-bath 100-110s that thaws, and then places under room temperature (18-20 ℃) condition and melts fully, activates in the nature seawater.
Further, the described essence of freezing is directly put into 37-40 ℃ of water-bath 100-110s and is thawed, during constantly shake cryopreservation tube.
Freezing smart the activation is specially, slide splashes into 100 μ L seawater, then add 1-2 μ L and freeze essence, inhale to beat and make its abundant mixing for 3-5 time, get immediately 3 visuals field, the quantity that detects the motion sperm accounts for the ratio of all sperms in the visual field, and the rate of motion of the rear sperm that thaws after testing and bright smart difference are not remarkable, do fast rectilinear motion more than 80%.
The present invention has following advantage:
1. adopt DMSO or the PG of high concentration to make antifreeze in the process of cryopreservation of the present invention, keep the better infiltrative while to reduce again toxicity;
2. added sucrose 34.2g/L in the anti frozen liquid of the present invention, 10% yolk (V/V) is as the outside protectant, and the plasma membrane of sperm is played a very good protection;
3. adopt the 2-5ml cryopreservation tube in the process of cryopreservation of the present invention, volume is larger, and the preservation sperm volume is large; Bright smart and anti frozen liquid concussion mixing 30s-1min directly puts into the programmed cooling instrument before the cooling, has saved the time, and is easy to operation; Simultaneously to be 3:1-4:1 improved more than ten doubly than the preservation ratio of 1:3-1:1 in the past to the ratio of sperm and anti frozen liquid, and the cryopreservation tube sperm storage amount of equal volume is large, has important production meaning.
4. when process of cryopreservation segmentation of the present invention is lowered the temperature, employing is with the cooling of-8--10 ℃/min speed, suitable speed is that sperm can fully dewater and can make again the frozen sperm degree of safety cross " dangerous temperature district " simultaneously, then drops into liquid nitrogen, and whole preservation process is no more than 15min; Freezing preservation cooling process is accurate, repeated good stability, and it is high to freeze the essence rear anabiosis rate that thaws, and rate of motion is higher than 80% after activating, and is not remarkable with the smart vigor state difference of aquatic foods;
5. after frozen sperm of the present invention takes out from liquid nitrogen, adopt 37 ℃-40 ℃ to thaw, can make the sperm fast speed cross the annealed zone, avoided again the too high damage that causes of local temperature simultaneously.
Embodiment
Embodiment 1:
1) in April, 2013 is on the occasion of turbot parent population reproduction period, in aquaculture station, Huang Island Jiangnan, the parent population cultivating workshop is pulled male parent population out from culturing pool, be positioned on the sponge, distilled water flushing gonopore three times, paper handkerchief is cleaned, and obtains gently the about 30ml of fresh semen of more than ten tail milters from backward front extruding belly, be stored in the clean centrifuge tube, lucifuge, the microscopic examination vigor is higher than 90%, places ice chest to take back the laboratory and carries out freezing preservation;
2) anti frozen liquid preparation is comprised of dilution, antifreeze, additive and distilled water, and configuration is placed on 0 ℃ of refrigerator precooling, and is stand-by; Wherein, dilution is: NaCl:8g/L, KCl:0.4g/L, MgSO
47H
2O:0.1g/L, MgCl
26H
2O:0.1g/L, Na
2HPO
42H
2O:0.06g/L, KH
2PO
4: 0.06g/L, NaHCO
3: 0.35g/L; The antifreeze final concentration is 60%DMSO(V/V); Additive: sucrose 34.2g/L, 10% yolk (V/V), described yolk by the egg protein isolate after and the gained that stirs.
The layoutprocedure of anti frozen liquid: at first dispose dilution with distilled water, take by weighing NaCl8g, KCl0.4g, MgSO
47H
2O0.1g, MgCl
26H
2O0.1g, Na
2HPO
42H
2O0.06g, KH
2PO
40.06g, NaHCO
30.35g it is for subsequent use then to be settled to 1L; Adopt the dilution that has disposed to dispose anti frozen liquid as basal liquid, be specially: get a beaker and add 60ml DMSO or PG, add sucrose 3.42g, yolk 10ml is settled to 100mL in the adding volumetric flask.
3) with above-mentioned bright smart bright smart with volume 3:1 or 4:1(with anti frozen liquid: ratio anti frozen liquid) is mixed, then (0 ℃) light shaking, abundant mixing 30s-1min in trash ice, divide to be filled in 2ml or the 5ml cryopreservation tube, the programmed cooling instrument is opened simultaneously, is chilled in advance 0 ℃;
4) cryopreservation tube is placed cooling instrument be cooled to-80 ℃ with-8 ℃/min rate of temperature fall, then be cooled to-120 ℃ with-10 ℃/min rate of temperature fall, balance 2 minutes, all cryopreservation tubes are poured in the foam box that fills liquid nitrogen, and then order is put into freezing storing box and is then put into the medium-term and long-term storage of liquid nitrogen (196 ℃) container one by one;
5) seminal fluid after above-mentioned freezing 2 weeks of preservation is thawed, choosing arbitrarily 3 cryopreservation tubes thaws, the front incubation chamber that fills liquid nitrogen that freezing storing box is placed first of thawing, then cryopreservation tube is taken out from box fast and put into 37-40 ℃ of water-bath, shake gently 100-110s, then place room temperature (18-20 ℃) to melting fully;
6) melt rear seminal fluid and activate with nature seawater above-mentioned, be specially, slide splashes into 100 μ L seawater, then add 1-2 μ L and freeze essence, suction is beaten and is made its abundant mixing for 3-5 time, gets immediately 3 visuals field, and the quantity that detects the motion sperm accounts for the ratio of all sperms in the visual field, rate of motion and the bright smart difference of rear sperm of thawing after testing is not remarkable
Activation is rear at microscopic examination thawn motility 80-90%, and the 80% above essence of freezing is all made fast rectilinear motion.
7) after activating freeze smart in average point-to-point speed, all not remarkable with bright smart difference on averaged curve movement velocity and the average path movement velocity.
Embodiment 2
1) with aquaculture station, Huang Island Jiangnan, the parent population cultivating workshop is pulled milter out from culturing pool on the occasion of turbot reproduction period 4-5 month in 2012, be positioned on the sponge, and distilled water flushing gonopore three times, paper handkerchief is cleaned, and obtains gently bright smart from backward front extruding belly.Gather altogether turbot more than 50 tails, amount to bright smart 70ml, microscopic examination vigor 75-90% places ice chest to go back to the laboratory and carries out freezing preservation;
2) anti frozen liquid preparation is comprised of dilution, antifreeze, additive and distilled water, and configuration is placed on 0 ℃ of refrigerator precooling, and is stand-by; Wherein, dilution is: NaCl:8g/L, KCl:0.4g/L, MgSO
47H
2O:0.1g/L, MgCl
26H
2O:0.1g/L, Na
2HPO
42H
2O:0.06g/L, KH
2PO
4: 0.06g/L, NaHCO
3: 0.35g/L; Antifreeze is the DMSO(V/V of final concentration 60%) or the PG(V/V of final concentration 60%); Additive: sucrose 34.2g/L, 10% yolk (V/V), described yolk is by egg protein isolate and the gained that stirs.
The layoutprocedure of anti frozen liquid: at first dispose dilution with distilled water, take by weighing NaCl8g, KCl0.4g, MgSO
47H
2O0.1g, MgCl
26H
2O0.1g, Na
2HPO
42H
2O0.06g, KH
2PO
40.06g, NaHCO
30.35g it is for subsequent use then to be settled to 1L; Adopt the dilution that has disposed to dispose anti frozen liquid as basal liquid, be specially: get a beaker and add 60ml DMSO or PG, add sucrose 3.42g, yolk 10ml is settled to 100mL in the adding volumetric flask.
3) with above-mentioned bright smart bright smart with volume 3:1-4:1(with anti frozen liquid: ratio anti frozen liquid) is mixed, then (0 ℃) light shaking, abundant mixing 30s-1min in trash ice, divide to be filled in 2ml or the 5ml cryopreservation tube, the programmed cooling instrument is opened simultaneously, is chilled in advance 0 ℃;
4) cryopreservation tube is placed cooling instrument be cooled to-80 ℃ with-8 ℃/min rate of temperature fall, then be cooled to-120 ℃ with-10 ℃/min rate of temperature fall, balance 2 minutes, all cryopreservation tubes are poured in the foam box that fills liquid nitrogen, and then order is put into freezing storing box and is then put into medium-term and long-term store (196 ℃) of liquid nitrogen container one by one;
5) freeze smart long-term frozen and preserve above-mentioned, in December, 2012, take more than 20 pipes away by general aquatic products Co., Ltd and freeze the pedestrian worker's insemination that progresses greatly, seed rearing.Defreezing method: cryopreservation tube taken out from box fast put into 37-40 ℃ of water-bath, shake gently 100-110s, then place room temperature 18-20 ℃ to melt fully;
6) melt rear seminal fluid and activate with nature seawater above-mentioned, at microscopic examination thawn motility 80-90%, and the 80% above essence of freezing is all made fast rectilinear motion after activating.
6) will freeze the smart artificial insemination that is used for, fertilization rate, incubation rate are all more than 80%, and weed survival rate is with bright smart in significant difference.
Claims (6)
1. the method for the freezing preservation of a turbot sperm high-efficiency ultralow temperature is characterized in that:
With the seminal fluid of turbot and anti frozen liquid by volume the ratio of 3:1-4:1 mix, process through the cooling of two steps after the mixing, put into liquid nitrogen (196 ℃) after the step balance, packing is preserved;
Anti frozen liquid is comprised of dilution, antifreeze, additive three parts
Wherein, dilution is NaCl8g/L, KCl0.4g/L, MgSO
47H
2O0.1g/L, MgCl
26H
2O0.1g/L, Na
2HPO
42H
2O0.06g/L, KH
2PO
40.06g/L, NaHCO
30.35g/L; Antifreeze is final concentration 60%(V/V) the DMSO(dimethyl sulfoxide (DMSO)) or final concentration 60%(V/V), propane diols (PG); Additive: the yolk (V/V) of sucrose 34.2g/L and final concentration 10%.
2. by the method for the freezing preservation of turbot sperm high-efficiency ultralow temperature claimed in claim 1, it is characterized in that: described anti frozen liquid is positioned over before use in 0 ℃ of refrigerator precooling and spends the night, and is stand-by.
3. by the method for the freezing preservation of turbot sperm high-efficiency ultralow temperature claimed in claim 1, it is characterized in that: the seminal fluid of described turbot through the anti frozen liquid mixed diluting places the cryopreservation tube of 2ml or 5ml.
4. press the method for the freezing preservation of turbot sperm high-efficiency ultralow temperature claimed in claim 1, it is characterized in that: the seminal fluid of described turbot and anti frozen liquid be (0 ℃) light shaking, abundant mixing 30s-1min in trash ice, then be cooled to-80 ℃ with-8 ℃/min, then be cooled to-120 ℃ with-10 ℃/min, balance is put into liquid nitrogen (196 ℃) after 2 minutes, packing is preserved.
5. press the method for the freezing preservation of turbot sperm high-efficiency ultralow temperature claimed in claim 1, it is characterized in that: the turbot that packing is stored in the liquid nitrogen is frozen capable the thawing of progressing greatly, concrete grammar is: the cryopreservation tube that will have a seminal fluid is directly put into the 37-40 ℃ of water-bath 100-110s that thaws, then place under room temperature (18-20 ℃) condition and melt fully, activate in the nature seawater.
6. by the method for the freezing preservation of turbot sperm high-efficiency ultralow temperature claimed in claim 1, it is characterized in that: the described essence of freezing is directly put into 37-40 ℃ of water-bath 100-110s and is thawed, during constantly shake cryopreservation tube.
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Cited By (9)
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| CN103891712A (en) * | 2014-04-01 | 2014-07-02 | 江苏省淡水水产研究所 | Ultralow-temperature cryopreservation method for channel catfish sperms |
| CN104894061A (en) * | 2015-05-29 | 2015-09-09 | 中国科学院海洋研究所 | Backcross artificial insemination method for cryopreserved paralichthys dentatus sperms and paralichthys olivaceus ova |
| CN105062954A (en) * | 2015-08-17 | 2015-11-18 | 宁波市海洋与渔业研究院 | Ultra-low temperature frozen mackerel semen thawing method |
| CN105532639A (en) * | 2015-12-18 | 2016-05-04 | 中国科学院海洋研究所 | Giant grouper sperm large-capacity efficient ultra-low-temperature cryopreservation method |
| CN107047540A (en) * | 2017-03-29 | 2017-08-18 | 华南农业大学 | A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution |
| CN108244099A (en) * | 2018-03-02 | 2018-07-06 | 山东省海洋生物研究院 | A kind of greenling sperm high-efficiency ultralow temperature freezing and storing method |
| CN108552161A (en) * | 2018-05-22 | 2018-09-21 | 华中农业大学 | A kind of the Ultra-cryofreezing preservation liquid and its Cryopreservation methods and applications of loach sperm |
| 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 |
| CN112715534A (en) * | 2021-03-01 | 2021-04-30 | 内江师范学院 | Efficient ultralow-temperature cryopreservation method for spring fish sperms |
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| CN103891712A (en) * | 2014-04-01 | 2014-07-02 | 江苏省淡水水产研究所 | Ultralow-temperature cryopreservation method for channel catfish sperms |
| CN104894061A (en) * | 2015-05-29 | 2015-09-09 | 中国科学院海洋研究所 | Backcross artificial insemination method for cryopreserved paralichthys dentatus sperms and paralichthys olivaceus ova |
| CN105062954A (en) * | 2015-08-17 | 2015-11-18 | 宁波市海洋与渔业研究院 | Ultra-low temperature frozen mackerel semen thawing method |
| CN105532639A (en) * | 2015-12-18 | 2016-05-04 | 中国科学院海洋研究所 | Giant grouper sperm large-capacity efficient ultra-low-temperature cryopreservation method |
| CN105532639B (en) * | 2015-12-18 | 2018-03-02 | 中国科学院海洋研究所 | A kind of sub- Large Copacity of epinephelus lanceolatus milt, the method for high-efficiency ultralow temperature freezen protective |
| CN107047540A (en) * | 2017-03-29 | 2017-08-18 | 华南农业大学 | A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution |
| CN107047540B (en) * | 2017-03-29 | 2020-12-29 | 华南农业大学 | Ultralow-temperature preservation method for seminal fluid of leiocassis longirostris and diluent thereof |
| CN108244099A (en) * | 2018-03-02 | 2018-07-06 | 山东省海洋生物研究院 | A kind of greenling sperm high-efficiency ultralow temperature freezing and storing method |
| CN108552161A (en) * | 2018-05-22 | 2018-09-21 | 华中农业大学 | A kind of the Ultra-cryofreezing preservation liquid and its Cryopreservation methods and applications of loach sperm |
| 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 |
| CN112715534A (en) * | 2021-03-01 | 2021-04-30 | 内江师范学院 | Efficient ultralow-temperature cryopreservation method for spring fish sperms |
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