CN103141472A - Ultra-low-temperature freezing preservation and activation method for Hong Kong oyster sperm - Google Patents
Ultra-low-temperature freezing preservation and activation method for Hong Kong oyster sperm Download PDFInfo
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Abstract
The invention discloses an ultra-low-temperature freezing preservation method for Hong Kong oyster sperm. The method comprises the following steps of collection, dilution and refrigeration of Hong Kong oyster sperm, and is characterized in that for the step of dilution, a diluent comprising a base liquid and an antifreeze agent is used for diluting and balancing the Hong Kong oyster sperm, for the refrigeration step, a programmed freezing manner is adopted for freezing, and finally the oyster sperm is preserved in liquid nitrogen. An activation method for the Hong Kong oyster sperm frozen and preserved by the method comprises the steps of defrosting and activating, wherein for the defrosting step, the sperm preserved in the liquid nitrogen is taken out and is completely defrosted in constant water being 35-55 DEG C, for the activating step, ammonia sea water with final mass concentration being 0.1563-0.6250%is added into recovered sperm. Due to the method, the rate of survival, vitality, fertility rate, and hatchability of the Hong Kong oyster sperm are improved, so that the breeding course of the Hong Kong oyster is accelerated, and the method is beneficial to the breeding of the Hong Kong oyster and large-scale breeding of fine breed, and has significance on preservation of Hong Kong oyster germplasm resources and conservation study of biodiversity.
Description
Technical field
The invention belongs to the sperm super-low temperature freezing technical field, particularly a kind of Hong Kong oyster sperm super-low temperature freezing preservation and Activiation method.
Background technology
Hong Kong oyster (
Crassostrea rivularis) be subordinate to lamellibranchiata, oyster order, Ostreidae.It is the common shellfish with economic worth of China.Hong Kong oyster is the main cultivation object of Guangxi mariculture industry, and ALONG GUANGXI COAST is the main original producton location of Hong Kong oyster.In recent years, Hong Kong oyster of cultivation occurs extensive dead, and output keeps falling, decline in benefits.Germplasm is degenerated and the seed shortage is the primary restraining factors of Hong Kong oyster culture industry development, and the research of carrying out Hong Kong oyster fine-variety breeding and scale seed breeding is extremely urgent.
At present sperm freezing technology in the mammal such as pig, ox, sheep, dog and the mankind is comparatively ripe, and the fish in aquatic animal, pteria martensii, Pacific oyster sperm freezing also have report, find following pertinent literature:
1. application number: 200710156398.8, denomination of invention: Cryopreservation of Boar Semen pipe and pig seminal fluid cryopreservation method thereof, the main points of its pig seminal fluid cryopreservation method are: the seminal fluid after the chill back after the pretreatment of pig seminal fluid and seminal fluid cooling processing is packed in the toroidal container of Cryopreservation of Boar Semen pipe of this invention, then the Cryopreservation of Boar Semen pipe is inserted on liquid nitrogen, stifling after several minutes, the medium-term and long-term preservation of direct plunge into Liquid Nitrogen.Advantage is: have the contact area that increases seminal fluid and liquid nitrogen in refrigerating process, accelerate semen freezing speed, reduce freezing damage to sperm, improve cold smart motility rate; Also have cost low, the operation box lunch, effect is high, guarantees the advantages such as conception rate.
2. application number: 200710043611.4, denomination of invention: quick freezing conservation method for bastard halibut spermatozoon, its technical scheme are with the natural sea-water dilution, with the dilute seawater that can not activate sperm as the dilution of preserving sperm; Adopting concentration is that 14g/100ml ethylene glycol is as freezant; With the cryogenic freezing protection liquid of dilution+antifreeze as bastard halibut spermatozoon, with the insulating box precooling of frozen solution at 4 ℃; Sperm evenly mixes in 1: 1 to 1: 3 ratio with protection liquid puts into centrifuge tube, and centrifuge tube is placed in-20 ℃ of balance 1min under the liquid nitrogen container mouth, then drops in liquid nitrogen fast and preserves.The advantage of this invention is easy and simple to handle, can the rapid saving bastard halibut spermatozoon.
3. application number: 201110258042.1, denomination of invention: oldwife milt is preserved freezant and the sub-store method of oldwife milt, its freezant by freeze-extender and antifreeze according to 9: the ratio 1(volume ratio) mixes, wherein the constituent content of freeze-extender is: NaCl:42-48mM, KHCO
3: 2.5-3.5mM, CaCl
2: 2.5-3.5mM, glucose: 35-45mM, Tris:1.5-2.5mM, glutathione: 1.5-2.5mM, bovine serum albumin: 1.5-2.5g/L, the pH value scope of described freeze-extender is: 6.7-6.9; Antifreeze in described freezant is methyl-sulfoxide (DMSO).And utilize this freezant to carry out the method that the sub-ultralow temperature of oldwife milt is preserved.This freezant and this store method, can long preservation oldwife milt, and the rear sperm viability that can guarantee to thaw still can reach more than 80%.
4. Lee's Yun, He Guizhen, Wang Pinhong; Pacific oyster sperm before and after ultralow temperature is preserved
(Crassostreagigas(Thunberg))Ultrastructural observation, Qingdao Marine University's journal, 2002,32(4): 526 ~ 532; Only mention application scanning Electronic Speculum and transmission electron microscopy, Pacific oyster sperm morphology and ultra microstructure before and after the research superfreeze.
Superfreeze is preserved, and refers under ultralow temperature (196 ~-198 ℃) condition all metabolic activities in the Inhibit sperm cell, and makes spermoblast by long preservation and a kind of Techniques of preserving of non-loss of activity.Spermoblast why can long preservation under condition of ultralow temperature, because in spermoblast, the chemical change in all metabolic processes is suppressed by ultralow temperature, namely along with the decline gradually of temperature, intracellular chemical reaction ability is also reducing gradually, when temperature dropped to certain limit, in cell, all chemical changes also just were in a kind of " time-out " state and make cell be able to long preservation; After the cell that low temperature is preserved is recovered in some way, has again the ability of survival.
The factor that affects refrigerating effect mainly comprises the selection of the selection of source, freeze-extender and the antifreezing agent of sperm, freezing and defreezing method.External pressure in the refrigerating process of sperm as the toxicity of cold shock, antifreezing agent, the formation of ice crystal, the change of osmotic pressure, all can cause the damage of sperm and function, and its degree of injury is decided by the tolerance of sperm self to extraneous pressure.The basal liquid of its freezing preservation of different plant species, freezeproof protectant, cool-down method, and recovery all variant.At present, not yet find about Hong Kong oyster sperm super-low temperature freezing and preserve and the report of Activiation method.
Summary of the invention
The objective of the invention is in order to solve the Techniques of preserving difficult problem of elite germplasm in the oyster fine-variety breeding of Hong Kong; provide a kind of Hong Kong oyster sperm super-low temperature freezing to preserve and Activiation method; Hong Kong oyster sperm survival rate, vigor, fertilization rate and incubation rate have been improved by the present invention; can accelerate the breeding process of Hong Kong oyster; for the development of Hong Kong oyster breeding from now on and the scale seed breeding of breeding are offered help, significant aspect the preservation of Hong Kong oyster germ plasm resource and biodiversity conservation research.
To achieve these goals, the present invention is achieved by the following technical solutions:
A kind of Hong Kong oyster sperm super-low temperature freezing store method comprises Hong Kong oyster sperm collection, dilution and freezing, described dilution be use the dilution that formed by basal liquid and antifreeze to Hong Kong oyster sperm dilute, balance; Described freezing be that to adopt programmed cooling method to carry out freezing, finally preserve in liquid nitrogen.
In the present invention, the collection of Hong Kong oyster sperm is also an important link, and the interindividual semen quality of same species has a great difference.Generally, freezing front semen quality is better, and the vigor of the rear sperm that thaws will be better.Therefore, selected Hong Kong oyster in the present invention, individual long 5 ~ 7cm, wide 3 ~ 5cm, high 10 ~ 11cm.
As further instruction, solution or normal saline solution that the above basal liquid is comprised of sodium citrate and glycine.
As preferably, the solution that the above sodium citrate and glycine form, its formula is: sodium citrate 0.1 ~ 0.3mol/L, glycine 0.2 ~ 0.4mol/L, surplus is sterile distilled water.
As preferably, the mass concentration of the above normal saline solution is 0.6 ~ 0.9%.
In the present invention, selected basal liquid and the concentration range that limits, Main Function is osmotic pressure and the pH value of optimizing Hong Kong oyster sperm environment of living in, for Hong Kong oyster sperm provides energy, prevent the loss of phosphatide in spermoblast and play a part antifreezing agent, prevent germ contamination, adapt to external environment to sperm and create favorable conditions.
As preferably, the above antifreeze is comprised of dimethyl sulfoxide (DMSO) and trehalose, and the volume content of dimethyl sulfoxide (DMSO) in dilution is 10%～14%, and trehalose content in dilution is 0.25～0.45mol/L.Because of the sperm of different plant species also different to ability to bear and its permeability of different antifreezing agent toxic actions, the present invention is through screening, combination and adjusting, antifreeze is optimized, can effectively reduce the freezing injury in the oyster sperm freezing process of Hong Kong, and toxic action is low, and is also less on the impact of Hong Kong oyster sperm recovery process.
As further instruction, the above dilution is to add in seminal fluid dilute at 20 ~ 80: 1 by volume.
As further instruction, the above balance be with after dilution and seminal fluid mixing under 3 ~ 5 ℃ of conditions balance 10 ~ 60min.Its objective is to guarantee that sperm has one section process that adapts to low temperature, simultaneously antifreeze is fully penetrated in the oyster spermoblast of Hong Kong and play a role, reduce, even avoided the cold shock damage of sperm, and then play freeze proof protective effect.
As further instruction, the above programmed cooling method is that the seminal fluid after dilution is down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min again, at-80 ℃ of balance 5min, at last it is taken out from the programmed cooling instrument and put in the cotton string bag, then drop in-196 ℃ ~-198 ℃ liquid nitrogen containers and preserve.In the refrigerating process of sperm, freezing rate can have influence on the osmotic pressure of the inside and outside solution of spermoblast and the balance of pH, is the deciding factor that superfreeze is preserved.Programmed cooling method is to utilize the programmed cooling instrument, according to the cooling process of design in advance, the residing environment of sperm is down to uniform temperature, then puts it in liquid nitrogen and preserves.This is the slower frozen mode of a kind of cooling rate.Cross slow cooling procedure the electrolyte concentration of solution is increased gradually, and when in the solution of sperm in high concentration, open-assembly time is long, can cause the destroyed and film of spermoblast membrane lipoprotein complex to be decomposed.When the cell membrane syneresis reaches critical minimum volume, can make again the cell permeability of the membrane produce irreversible damage, that the solute that originally can not see through film become permeability and then cause cellular damage or death.Cooling velocity is slower, increases caused osmotic pressure by electrolyte concentration and damages just larger; If the raising cooling rate can cause the formation of intracellular ice crystal and causes damage, cooldown rate is faster, and the quantity that ice crystal forms is more, and damage is just larger.Therefore, freezing procedure preferred for this invention is that sperm super-low temperature is preserved the scope that obtains best rate of temperature fall.
Hong Kong as above oyster activation of spermatozoa method comprises and thaws and activate step, and described thawing is liquid nitrogen to be preserved sperm take out, and thaws to melting fully 35～55 ℃ of waters bath with thermostatic control; Described activation is that to add whole mass concentration after the recovery of thawing in sperm be 0.1563～0.6250 ‰ ammonia seawater, and namely the ammonia seawater mixes with seminal fluid that in rear seminal fluid, the ammonia mass content is 0.1563～0.6250 ‰, makes its recovery fertility.After activating, sperm viability is 56% ~ 77%, and survival rate is 70% ~ 87%.
In the present invention, after the oyster activation of spermatozoa of Hong Kong, carry out subsequently sperm quality and detect, the dynamic and static feature of sperm is carried out quantitative analysis, thereby sperm quality is made thoroughly evaluating, obtain that survival rate is high, energetic, fertilization rate is high Hong Kong oyster sperm.It is to utilize Computer Recognition Technology and image processing techniques that described sperm quality detects, dynamic and static nature to sperm carries out comprehensive quantitative analysis, detects movement locus, movement rate, distribution, sperm profile, sperm quantity, viscosity and the vigor of sperm.
Compared with prior art, the invention has the beneficial effects as follows:
1. the selected dilution of the present invention, solution or normal saline solution that basal liquid is comprised of sodium citrate and glycine, antifreeze is comprised of dimethyl sulfoxide (DMSO) and trehalose, its toxic action is low, and optimized osmotic pressure and the pH value of Hong Kong oyster sperm environment of living in, for Hong Kong oyster sperm provides energy, prevented the loss of phosphatide in spermoblast and play a part antifreezing agent, prevent germ contamination, adapt to external environment to sperm and create favorable conditions.
2. the present invention adopts programmed cooling method, selected best rate of temperature fall, both reduced that cooling rate is too high to be caused the formation of intracellular ice crystal and cause damage, and also avoided the too low destroyed and film of spermoblast membrane lipoprotein complex that causes of cooling rate to be decomposed.
3. the present invention preserves sperm with liquid nitrogen and thaws to melting fully 35～55 ℃ of waters bath with thermostatic control, then to add whole mass concentration after the recovery in sperm be 0.1563～0.6250 ‰ ammonia seawater, and its cellular damage rate is minimum, and activity ratio is the highest.
4. the present invention can superfreeze preserve Hong Kong oyster sperm, and after recovery, its sperm survival rate is high, energetic, can apply to needs of production, and can reach the purpose of taking at any time; Having broken different plant species is restricted to distant hybridization mating season, cultivates fine quality new way is provided; For wild germplasm protection of resources, breeding germplasm are preserved, genetic diversity conservation provides technical support.
Embodiment
The present invention is described in further detail below in conjunction with embodiment, but embodiments of the present invention are not limited to the scope that embodiment represents.
Embodiment 1:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 5cm, wide 3cm, high 10cm Hong Kong oyster to dissect, choose through being accredited as 5 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution, the content of sodium citrate 0.1124mol/L wherein, glycine content 0.2667 mol/L, trehalose 0.25 mol/L, dimethyl sulfoxide (DMSO) 10%(v/v), surplus is sterile distilled water;
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:80 dilution, are kept in 1.5ml plastics cryovial, after 4 ℃ of refrigerator balance 30min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-198 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 35 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.1563 ‰ ammonia seawater, begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 56.88～68.14%, and survival rate is 70.69～79.58%.
Embodiment 2:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 6cm, wide 4cm, high 10.5cm Hong Kong oyster to dissect, choose through being accredited as 4 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution, the content of sodium citrate 0.1mol/L wherein, glycine content 0.4mol/L, trehalose 0.30 mol/L, dimethyl sulfoxide (DMSO) 12%(v/v), surplus is sterile distilled water;
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:60 dilution, are kept in 1.5ml plastics cryovial, after 3 ℃ of refrigerator balance 10min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-196 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 45 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.2489 ‰ ammonia seawater.Begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 55.72～67.65%, and survival rate is 69.88～78.40%.
Embodiment 3:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 7cm, wide 5cm, high 11cm Hong Kong oyster to dissect, choose through being accredited as 5 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution, the content of sodium citrate 0.3mol/L wherein, glycine content 0.2mol/L, trehalose 0.45 mol/L, dimethyl sulfoxide (DMSO) 14%(v/v), surplus is sterile distilled water;
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:20 dilution, are kept in 1.5ml plastics cryovial, after 5 ℃ of refrigerator balance 60min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-197 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 45 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.3945 ‰ ammonia seawater.Begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 58.04～71.23%, and survival rate is 71.54～80.02%.
Embodiment 4:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 6cm, wide 3cm, high 10cm Hong Kong oyster to dissect, choose through being accredited as 4 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution protection liquid, wherein sodium chloride is 0.8%(m/m), trehalose 0.25 mol/L, dimethyl sulfoxide (DMSO) 10%(v/v).
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:80 dilution, are kept in 1.5ml plastics cryovial, after 4 ℃ of refrigerator balance 30min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-198 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 35 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.5466 ‰ ammonia seawater.Begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 60.91～76.07%, and survival rate is 77.61～86.92%.
Embodiment 5:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 6cm, wide 3cm, high 10cm Hong Kong oyster to dissect, choose through being accredited as 4 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution protection liquid, wherein sodium chloride is 0.6%(m/m), trehalose 0.40 mol/L, dimethyl sulfoxide (DMSO) 13%(v/v), surplus is sterile distilled water.
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:80 dilution, are kept in 1.5ml plastics cryovial, after 4 ℃ of refrigerator balance 30min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-198 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 35 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.6250 ‰ ammonia seawater.Begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 59.81～75.85%, and survival rate is 78.12～87.98%.
Embodiment 6:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 6cm, wide 3cm, high 10cm Hong Kong oyster to dissect, choose through being accredited as 4 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution protection liquid, wherein sodium chloride is 0.9%(m/m), trehalose 0.45 mol/L, dimethyl sulfoxide (DMSO) 10%(v/v).
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:80 dilution, are kept in 1.5ml plastics cryovial, after 4 ℃ of refrigerator balance 30min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-198 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 35 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.5421 ‰ ammonia seawater.Begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 61.27～77.46%, and survival rate is 78.61～87.72%.
Embodiment 7:
1) sperm collection: choose Hong Kong oyster of waters, town, Guangxi Qinzhou gantry cultivation, select individual long 7cm, wide 5cm, high 11cm Hong Kong oyster to dissect, choose through being accredited as 3 of male individualities and use after with its sperm mixing.
2) dilution: configuration 100ml dilution protection liquid; D-Hank ' s liquid (chloride containing potassium 0.4g in 1 liter of solution wherein; potassium dihydrogen phosphate 0.06g; sodium chloride 8.00g; sodium bicarbonate 0.35g, 7 water sodium hydrogen phosphate 0.09g, phenol red 0.01g); trehalose 0.25 mol/L, dimethyl sulfoxide (DMSO) 10%(v/v).
3) programmed cooling instrument programming: be down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, then be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min, at-80 ℃ of balance 5min.
4) seminal fluid and dilution according to volume ratio 1:80 dilution, are kept in 1.5ml plastics cryovial, after 4 ℃ of refrigerator balance 30min, namely put into the programmed cooling instrument start program cooling of having established program.
5) be kept at freezing preservation in-198 ℃ of liquid nitrogen containers after EP (end of program), thaw fully in 35 ℃ of waters bath with thermostatic control after preserving 48h, add whole mass concentration after the recovery in sperm and be 0.6250 ‰ ammonia seawater.Begin to detect.
6) utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.Detect analysis result: sperm viability is 44-47.28%, and survival rate is 71.86-72.58%.
Embodiment 7 is corresponding experiment, by embodiment 1 ~ 6 and embodiment 7 contrasts, can find out that the dilution of this method can improve sperm viability and survival rate.
Claims (9)
1. Hong Kong oyster sperm super-low temperature freezing store method, comprise Hong Kong oyster sperm collection, dilution and freezing, it is characterized in that: described dilution be use the dilution that formed by basal liquid and antifreeze to Hong Kong oyster sperm dilute, balance; Described freezing be that to adopt programmed cooling method to carry out freezing, finally preserve in liquid nitrogen.
2. Hong Kong according to claim 1 oyster sperm super-low temperature freezing store method, is characterized in that: solution or normal saline solution that described basal liquid is comprised of sodium citrate and glycine.
3. Hong Kong according to claim 2 oyster sperm super-low temperature freezing store method, it is characterized in that: the solution that described sodium citrate and glycine form, its formula is: sodium citrate 0.1 ~ 0.3mol/L, and glycine 0.2 ~ 0.4mol/L, surplus is sterile distilled water.
4. Hong Kong according to claim 2 oyster sperm super-low temperature freezing store method, it is characterized in that: the mass concentration of described normal saline solution is 0.6 ~ 0.9%.
5. the described Hong Kong of any one oyster sperm super-low temperature freezing store method according to claim 1-4, it is characterized in that: described antifreeze is comprised of dimethyl sulfoxide (DMSO) and trehalose, the volume content of dimethyl sulfoxide (DMSO) in dilution is 10%～14%, and trehalose content in dilution is 0.25～0.45mol/L.
6. Hong Kong according to claim 5 oyster sperm super-low temperature freezing store method is characterized in that: described dilution is to add in seminal fluid dilute at 20 ~ 80: 1 by volume.
7. Hong Kong according to claim 5 oyster sperm super-low temperature freezing store method is characterized in that: described balance be with after dilution and seminal fluid mixing under 3 ~ 5 ℃ of conditions balance 10 ~ 60min.
According to claim 1-4 and 6-7 in any one described Hong Kong oyster sperm super-low temperature freezing store method, it is characterized in that: described programmed cooling method is that the seminal fluid after dilution is down to-20 ℃ from 4 ℃ of rate of temperature fall with-5 ℃/min, at-20 ℃ of balance 5min, be down to-80 ℃ from-20 ℃ of speed with-10 ℃/min again, at-80 ℃ of balance 5min, take out at last to drop in-196 ℃ ~-198 ℃ liquid nitrogen containers and preserve.
9. Hong Kong as described in any one in claim 1 ~ 8 oyster activation of spermatozoa method, comprise and thaw and activate step, it is characterized in that: described thawing is liquid nitrogen to be preserved sperm take out, and thaws to melting fully 35～55 ℃ of waters bath with thermostatic control; Described activation is that to add whole mass concentration after recovery in sperm be 0.1563～0.6250 ‰ ammonia seawater.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103704204A (en) * | 2014-01-06 | 2014-04-09 | 中国科学院海洋研究所 | Ultralow-temperature freezing preservation method of pacific oyster embryos |
| CN103749357A (en) * | 2014-01-08 | 2014-04-30 | 中国科学院南海海洋研究所 | Method of producing seeds of Crassostrea hongkongensis by using gill aperture freeness as marker |
| CN104145944A (en) * | 2014-08-25 | 2014-11-19 | 山东省海洋生物研究院 | Ultralow-temperature cryopreservation and activation method of sperm of scapharca broughtonii sckrenck |
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| CN114208730A (en) * | 2021-12-10 | 2022-03-22 | 宁德师范学院 | Cryopreservation and recovery method for maintaining sperm insemination performance of large yellow croaker and application |
| 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 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103704204A (en) * | 2014-01-06 | 2014-04-09 | 中国科学院海洋研究所 | Ultralow-temperature freezing preservation method of pacific oyster embryos |
| CN103704204B (en) * | 2014-01-06 | 2016-04-06 | 中国科学院海洋研究所 | A kind of Pacific oyster embryo cryopreservation method |
| CN103749357A (en) * | 2014-01-08 | 2014-04-30 | 中国科学院南海海洋研究所 | Method of producing seeds of Crassostrea hongkongensis by using gill aperture freeness as marker |
| CN104145944A (en) * | 2014-08-25 | 2014-11-19 | 山东省海洋生物研究院 | Ultralow-temperature cryopreservation and activation method of sperm of scapharca broughtonii sckrenck |
| CN105123566A (en) * | 2015-07-23 | 2015-12-09 | 新疆农业大学 | Hucho taimen sperm activation solution and preparation method thereof |
| CN112806353A (en) * | 2020-12-30 | 2021-05-18 | 西南大学 | Method for preserving and recovering sturgeon sperms |
| CN114208730A (en) * | 2021-12-10 | 2022-03-22 | 宁德师范学院 | Cryopreservation and recovery method for maintaining sperm insemination performance of large yellow croaker and application |
| 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 |
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