CN112970742A - Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms - Google Patents
Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms Download PDFInfo
- Publication number
- CN112970742A CN112970742A CN202110220020.XA CN202110220020A CN112970742A CN 112970742 A CN112970742 A CN 112970742A CN 202110220020 A CN202110220020 A CN 202110220020A CN 112970742 A CN112970742 A CN 112970742A
- Authority
- CN
- China
- Prior art keywords
- phoxinus
- tinctorius
- freezing
- solution
- ultralow
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000145619 Phoxinus Species 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 16
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000007710 freezing Methods 0.000 claims abstract description 28
- 239000007788 liquid Substances 0.000 claims abstract description 18
- 238000001816 cooling Methods 0.000 claims abstract description 17
- 239000003085 diluting agent Substances 0.000 claims abstract description 15
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 14
- 238000004321 preservation Methods 0.000 claims abstract description 12
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000010902 straw Substances 0.000 claims description 23
- 239000000243 solution Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims description 20
- 210000000582 semen Anatomy 0.000 claims description 17
- 241000251468 Actinopterygii Species 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000011259 mixed solution Substances 0.000 claims description 12
- 241000209140 Triticum Species 0.000 claims description 11
- 235000021307 Triticum Nutrition 0.000 claims description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 230000019100 sperm motility Effects 0.000 claims description 10
- 229960003080 taurine Drugs 0.000 claims description 10
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 9
- 230000002528 anti-freeze Effects 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 229910052564 epsomite Inorganic materials 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000012466 permeate Substances 0.000 claims description 5
- 239000003223 protective agent Substances 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- 239000007798 antifreeze agent Substances 0.000 claims description 4
- 238000007598 dipping method Methods 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 230000003213 activating effect Effects 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000011002 quantification Methods 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 6
- 230000001488 breeding effect Effects 0.000 abstract description 6
- 238000009360 aquaculture Methods 0.000 abstract description 3
- 244000144974 aquaculture Species 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000002068 genetic effect Effects 0.000 abstract description 2
- 230000008014 freezing Effects 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000002577 cryoprotective agent Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000010257 thawing Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 240000006891 Artemisia vulgaris Species 0.000 description 1
- 235000003261 Artemisia vulgaris Nutrition 0.000 description 1
- 241001500232 Oxycephalus Species 0.000 description 1
- 241001125847 Tinca Species 0.000 description 1
- 241001125862 Tinca tinca Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000012214 genetic breeding Methods 0.000 description 1
- 210000004392 genitalia Anatomy 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to the technical field of aquaculture, and particularly relates to a high-efficiency ultralow-temperature cryopreservation method for phoxinus tinctorius sperms. Mixing the sperm of phoxinus xinus with the anti-freezing solution according to the volume ratio of 1:40, performing programmed cooling treatment after mixing, and finally adding into liquid nitrogen (-196 ℃), subpackaging and storing; the anti-freezing liquid consists of diluent and anti-freezing agent. The frozen sperm obtained by the invention has important significance for artificial breeding, germplasm preservation, genetic diversity protection, sustainable culture and the like of the phoxinus tinctorius.
Description
Technical Field
The invention belongs to the technical field of aquaculture, and particularly relates to a high-efficiency ultralow-temperature cryopreservation method for phoxinus tinctorius sperms.
Technical Field
The tench (rhynchophoric prises oxycephalus), also called mugwort, is a cold water small-sized cyprinid fish widely distributed in east asia, mainly inhabits upstream stream or mountain stream with high altitude, low water temperature, high dissolved oxygen, certain water flow speed and clear water quality, and is a common species or a dominant species of mountain stream in Sichuan province. The high requirement of the phoxinus acuminata on the inhabiting environment makes the phoxinus acuminata have the characteristics of tender meat and delicious taste, less interpupillary bones and high nutritional value, is deeply loved by wide consumers, and has the market price of up to 100 yuan/jin. The phoxinus tinctorius sold in the market at present mainly comes from natural populations, but the phoxinus tinctorius has small natural population quantity, low breeding investment and weak ecological tolerance, is particularly sensitive and fragile to abnormal temperature change, and is extremely easy to endanger and extinct in the face of human interference and environmental change. Therefore, the research and development of artificial breeding technology of phoxinus xinus is urgent, no matter based on the development and popularization of new species of native economic fish, or the protection of natural population. The ultralow temperature preservation of the fish semen has important significance in genetic breeding and germplasm resource preservation, can realize the long-distance transportation of germplasm, and is beneficial to the breeding of fish and the protection of gene diversity. The ultra-low temperature freezing technology for tinca sperm is urgently required for artificial breeding and genetic diversity protection.
Disclosure of Invention
The invention aims to provide a high-efficiency ultralow-temperature cryopreservation method for sperms of phoxinus acuminata.
In order to achieve the purpose, the invention adopts the technical scheme that:
mixing the semen of phoxinus acuminata with the anti-freezing solution according to the volume ratio of 1:40, performing step-by-step cooling treatment by a programmed cooling instrument, and finally adding into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreeze solution comprises diluent and antifreeze agent, wherein the diluent is NaCl 9.00g/L, KCl 0.42g/L, CaCl2 0.24g/L，MgSO4·7H2O 0.15g/L，NaHCO30.20g/L，NaH2PO30.05g/L and 4.5g/L taurine solution; the cryoprotectant was 35% (final concentration of cryoprotectant; V/V) DMSO (dimethyl sulfoxide).
The diluent is prepared by taking 9.00g of NaCl, 0.42g of KCl and CaCl2 0.24g，MgSO4·7H2O 0.15g，NaHCO3 0.20g，NaH2PO3Adding 0.05g of taurine and 4.5g of taurine into distilled water for full dissolution, and then putting into a quantitative bottle for quantification to 1L to obtain the taurine; the anti-freezing solution is prepared by adding 350mLDMSO (dimethyl sulfoxide) into 650mL of prepared diluent and fully and uniformly mixing.
Selecting 0.5mL of wheat tube, adding 500 μ L of mixed solution of the fine fish tip and the anti-freezing solution which are uniformly mixed according to the volume ratio of 1:40 in advance for 5min into each tube, quickly dipping PVC plastic dissolved by tetrahydrofuran at the opening of the wheat tube for 1-2 seconds, and placing the mixture into a refrigerator at 4 ℃ for precooling for 1min to ensure that the anti-freezing protective agent fully permeates into cells.
And (3) putting the subpackaged mixed solution into a programmed cooling instrument, cooling to-100 ℃ according to a cooling program of-25 ℃/min, and quickly putting into liquid nitrogen for preservation.
When in unfreezing, the straws are fished out of the liquid nitrogen by using tweezers, then the straws are quickly put into a 15 ℃ constant-temperature water bath pot for unfreezing in water bath for 10-15 seconds, the straws are gently shaken to be uniformly heated, and the straws are quickly taken out after unfreezing.
Cutting off both ends of the straw by scissors, allowing the semen mixture to flow into a glass slide, adding Actifish sperm motility activating solution to activate the sperm, and performing microscopic examination on the sperm motility.
Compared with the prior art, the invention has the following beneficial effects:
firstly, DMSO is used as an antifreeze agent in the process of cryopreservation, so that the toxicity is reduced while better permeability is kept;
secondly, 0.5mL of straw is adopted in the freezing preservation process, so that the permeability is good, the temperature change is sensitive, and the cooling rate can be accurately controlled;
mixing the antifreeze protective agent with the diluent to prepare a mixed solution before cooling, so that the time is saved, and the operation is simple and easy;
and fourthly, 0.5mL of wheat pipe is selected, 500 microliter of mixture of the phoxinus cuspidata semen and the antifreeze liquid which are uniformly mixed according to the volume ratio of 1:40 is added into each pipe 5min in advance, the PVC plastic dissolved by tetrahydrofuran is quickly dipped at the opening of the wheat pipe for 1-2 seconds, the wheat pipe can be quickly and effectively blocked, and the anti-freezing agent is nontoxic and harmless, fully permeates into cells to play a role in protection, and has important production significance.
Fifthly, when the freezing preservation process is cooled in a segmented manner, the subpackaged semen is put into a program cooling instrument, then is cooled to-100 ℃ according to a cooling program of-25 ℃/min, and is then quickly put into liquid nitrogen for preservation until activation, the proper cooling speed is the key point that the semen can be fully dehydrated, and the frozen semen can safely pass through a dangerous temperature zone, and the whole preservation process is short in time; the freezing preservation and cooling procedure is simple and easy to operate, the repeatability and stability are good, the recovery rate of frozen semen after thawing is high, the movement rate after activation is higher than 95%, and the activity difference with fresh semen is not obvious;
taking out the frozen sperm from the liquid nitrogen, fishing out the straw from the liquid nitrogen by using forceps during thawing, quickly putting the straw into a 15 ℃ constant-temperature water bath kettle for thawing for 10-15 seconds in a water bath, slightly shaking to ensure that the temperature is uniformly heated, and quickly taking out the frozen sperm after thawing, so that the sperm can quickly pass through a recrystallization zone, and the structural damage of the sperm caused by overhigh local temperature is avoided.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the invention thereto.
Example 1:
(1) in 1 month of 2020, 30 male fish tails were collected from Daxiong aquaculture companies in Luzhou, Sichuan province. The weight is 10g-15g, parent fish is cultivated at academy of Nei river until the male fish at 12 tails in 3 middle of the month is mature, and semen flows out by lightly pressing the abdomen with hands. Placing the mixture on a dry gauze, washing the genital pore with distilled water for three times until no urine and mucus are left, wiping the mixture with clean toilet paper, slightly pressing the mixture on the abdomen along the direction from the fish head to the fish tail, and collecting the uncontaminated semen in a 2mL centrifuge tube. Detecting sperm motility rate with computer-assisted sperm analysis system (CASA), and selecting sample with motility rate higher than 95% for cryopreservation.
(2) Preparing an anti-freezing solution, which consists of a diluent, an antifreeze and distilled water, and placing the prepared anti-freezing solution in a refrigerator for precooling at 0 ℃ for later use; wherein, the diluent is: NaCl 9.00g/L, KCl 0.42g/L, CaCl2 0.24g/L，MgSO4·7H2O 0.15g/L，NaHCO3 0.20g/L，NaH2PO30.05g/L and 4.5g/L taurine solution; the cryoprotectant concentration was 35% DMSO (V/V).
The preparation process of the anti-freezing liquid comprises the following steps: and (3) adding 350mL of prepared diluent 650mL into dimethyl sulfoxide (DMSO), and fully and uniformly mixing to obtain the product.
(3) Selecting 0.5mL of wheat tube, adding 500 μ L of mixed solution of the fine fish tip and the anti-freezing solution which are uniformly mixed according to the volume ratio of 1:40 in advance for 5min into each tube, quickly dipping PVC plastic dissolved by tetrahydrofuran at the opening of the wheat tube for 1-2 seconds, and placing the mixture into a refrigerator at 4 ℃ for precooling for 1min to ensure that the anti-freezing protective agent fully permeates into cells.
(4) And (3) putting the subpackaged mixed solution into a programmed cooling instrument, cooling to-100 ℃ according to a cooling program of-25 ℃/min, and quickly putting into liquid nitrogen for preservation.
(5) When in unfreezing, the straws are fished out of the liquid nitrogen by using tweezers, then the straws are quickly put into a 15 ℃ constant-temperature water bath pot for unfreezing in water bath for 10-15 seconds, the straws are gently shaken to be uniformly heated, and the straws are quickly taken out after unfreezing.
(6) Cutting off both ends of the straw by scissors, allowing the semen mixed solution to flow into a glass slide, adding Actifish sperm motility activating solution to absorb semen to activate sperm, and performing microscopic examination on the sperm motility. The indexes such as sperm motility rate are calculated by a Computer Assisted Sperm Analysis (CASA) system, and the frozen sperm motility rate (92.33% +/-2.56%) is detected by a microscope after activation and is close to the fresh sperm level.
Example 2
(1) In 2020, 6 positive-value phoxinus acuminata are bred in a busy season, 40 male fishes (with the weight of 12g-17g) are fished out from a parent fish breeding pond of three Toplongyuancun villages in Guanyuan city, Sichuan province, placed on a sponge, washed with distilled water for three times to clean reproductive holes, wiped with a paper towel, and lightly squeezed from the back to the front to obtain fresh extract. Collecting 50 phoxinus rhynchophylla tails and counting 10mL of fresh essence, wherein the microscopic activity is higher than 95%, and placing the phoxinus rhynchophylla tails in an ice box and returning the phoxinus rhynchophylla tails to a laboratory for freezing storage;
(2) preparing an anti-freezing solution, which consists of a diluent, an antifreeze and distilled water, and placing the prepared anti-freezing solution in a refrigerator for precooling at 0 ℃ for later use; wherein, the diluent is: NaCl 9.00g/L, KCl 0.42g/L, CaCl2 0.24g/L，MgSO4·7H2O 0.15g/L，NaHCO3 0.20g/L，NaH2PO30.05g/L and 4.5g/L taurine solution; the cryoprotectant concentration was 35% DMSO (V/V).
The preparation process of the anti-freezing liquid comprises the following steps: and (3) adding 350mL of prepared diluent 650mL into dimethyl sulfoxide (DMSO), and fully and uniformly mixing to obtain the product.
(3) Selecting 0.5mL of wheat tube, adding 500 μ L of mixed solution of the fine fish tip and the anti-freezing solution which are uniformly mixed according to the volume ratio of 1:40 in advance for 5min into each tube, quickly dipping PVC plastic dissolved by tetrahydrofuran at the opening of the wheat tube for 1-2 seconds, and placing the mixture into a refrigerator at 4 ℃ for precooling for 1min to ensure that the anti-freezing protective agent fully permeates into cells.
(4) And (3) putting the subpackaged mixed solution into a programmed cooling instrument, cooling to-100 ℃ according to a cooling program of-25 ℃/min, and quickly putting into liquid nitrogen for preservation.
(5) When in unfreezing, the straws are fished out of the liquid nitrogen by using tweezers, then the straws are quickly put into a 15 ℃ constant-temperature water bath pot for unfreezing in water bath for 10-15 seconds, the straws are gently shaken to be uniformly heated, and the straws are quickly taken out after unfreezing.
(6) The two ends of the straw are cut off by scissors, and the semen mixed solution flows into the glass slide to examine the vitality of the semen mixed solution under the microscope. And (3) calculating indexes such as sperm motility rate by using a Computer Assisted Sperm Analysis (CASA) system, and detecting that the sperm freezing motility rate is more than or equal to 93 percent and is close to the level of fresh sperm by using a microscope after activation.
Claims (6)
1. A high-efficiency ultralow-temperature cryopreservation method for phoxinus tinctorius sperms is characterized in that:
mixing the semen of phoxinus acuminata with the anti-freezing solution according to the volume ratio of 1:40, performing step-by-step cooling treatment by a programmed cooling instrument, and finally adding into liquid nitrogen (-196 ℃), subpackaging and storing; the antifreeze solution comprises diluent and antifreeze agent, wherein the diluent is NaCl 9.00g/L, KCl 0.42g/L, CaCl20.24g/L，MgSO4·7H2O 0.15g/L，NaHCO30.20g/L，NaH2PO30.05g/L and 4.5g/L taurine solution; the antifreeze agent is 35% (V/V) DMSO (dimethyl sulfoxide).
2. The high-efficiency ultralow-temperature cryopreservation method for spermatozoa of phoxinus tinctorius according to claim 1, which is characterized in that: the diluent is prepared by taking 9.00g of NaCl, 0.42g of KCl and CaCl20.24g，MgSO4·7H2O 0.15g，NaHCO30.20g，NaH2PO3Adding 0.05g of taurine and 4.5g of taurine into distilled water for full dissolution, and then putting into a quantitative bottle for quantification to 1L to obtain the taurine; the anti-freezing solution is prepared by adding 350mLDMSO (dimethyl sulfoxide) into 650mL of prepared diluentMixing to obtain the final product.
3. The high-efficiency ultralow-temperature cryopreservation method for spermatozoa of phoxinus tinctorius according to claim 1, which is characterized in that: selecting 0.5mL of wheat tube, adding 500 μ L of mixed solution of the fine fish tip and the anti-freezing solution which are uniformly mixed according to the volume ratio of 1:40 in advance for 5min into each tube, quickly dipping PVC plastic dissolved by tetrahydrofuran at the opening of the wheat tube for 1-2 seconds, and placing the mixture into a refrigerator at 4 ℃ for precooling for 1min to ensure that the anti-freezing protective agent fully permeates into cells.
4. The method for the efficient ultralow-temperature cryopreservation of spermatozoa of phoxinus tinctorius according to claim 1, which is characterized in that: and (3) putting the subpackaged mixed solution into a programmed cooling instrument, cooling to-100 ℃ according to a cooling program of-25 ℃/min, and quickly putting into liquid nitrogen for preservation.
5. The high-efficiency ultralow-temperature cryopreservation method for spermatozoa of phoxinus tinctorius according to claim 1, which is characterized in that: when in unfreezing, the straws are fished out of the liquid nitrogen by using tweezers, then the straws are quickly put into a 15 ℃ constant-temperature water bath pot for unfreezing in water bath for 10-15 seconds, the straws are gently shaken to be uniformly heated, and the straws are quickly taken out after unfreezing.
6. The high-efficiency ultralow-temperature cryopreservation method for spermatozoa of phoxinus tinctorius according to claim 1, which is characterized in that: cutting off both ends of the straw by scissors, allowing the semen mixture to flow into a glass slide, adding Actifish sperm motility activating solution to activate the sperm, and performing microscopic examination on the sperm motility.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110220020.XA CN112970742A (en) | 2021-02-26 | 2021-02-26 | Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110220020.XA CN112970742A (en) | 2021-02-26 | 2021-02-26 | Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112970742A true CN112970742A (en) | 2021-06-18 |
Family
ID=76351219
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110220020.XA Pending CN112970742A (en) | 2021-02-26 | 2021-02-26 | Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112970742A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116210627A (en) * | 2023-03-13 | 2023-06-06 | 内江师范学院 | Artificial induced spawning and hatching method for tinca |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703039A (en) * | 2009-11-10 | 2010-05-12 | 淮海工学院 | Sperm cryopreservation method of Charybdisjaponica |
| 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 |
| CN109362715A (en) * | 2018-12-24 | 2019-02-22 | 内江师范学院 | A kind of expanded letter sand loach semen cryopreservation liquid and cryopreservation method |
-
2021
- 2021-02-26 CN CN202110220020.XA patent/CN112970742A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703039A (en) * | 2009-11-10 | 2010-05-12 | 淮海工学院 | Sperm cryopreservation method of Charybdisjaponica |
| 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 |
| CN109362715A (en) * | 2018-12-24 | 2019-02-22 | 内江师范学院 | A kind of expanded letter sand loach semen cryopreservation liquid and cryopreservation method |
Non-Patent Citations (3)
| Title |
|---|
| 成都德麟科技有限公司: "《Actifish淡水鱼精子活力激活液》", 12 May 2016 * |
| 桑润滋: "《动物繁殖生物技术》", 30 June 2002, 中国农业出版社 * |
| 陈炜: "《伊甸园的众生：生物学与高新技术》", 30 November 1999, 湖北科学技术出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116210627A (en) * | 2023-03-13 | 2023-06-06 | 内江师范学院 | Artificial induced spawning and hatching method for tinca |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kurokura et al. | Cryopreservation of carp sperm | |
| CN102077823B (en) | Method for cryopreservation of semen of siniperca scherzeri and artificial insemination | |
| CN104488853B (en) | A kind of ox embryo vitrifying freeze gets rid of pipe and thaws and directly transplanting method | |
| Uemura et al. | Survival of carnation (Dianthus caryophyllus L.) shoot apices frozen to the temperature of liquid nitrogen | |
| CN104521943A (en) | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen | |
| CN112741078B (en) | Hexagrammos otakii sperm productive cryopreservation method | |
| CN108244099A (en) | A kind of greenling sperm high-efficiency ultralow temperature freezing and storing method | |
| CN104145944B (en) | A kind of stalwart blood clam sperm super-low temperature freezing is preserved and Activiation method | |
| CN103348966A (en) | Method for efficient ultralow temperature cryopreservation of turbot sperms | |
| CN103314949B (en) | Ultralow temperature cryopreservation method for preserving sperm of Pacific cod | |
| CN110881460A (en) | Cryopreservation method for sperm of Oplophorus argus | |
| CN112970742A (en) | Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms | |
| CN102763591A (en) | Atrac tylodes macrocephala koidz conservation method and re-cultivating method | |
| CN106818708B (en) | Ultralow temperature cryoprotectant for epinephelus coioides semen and preservation method thereof | |
| Bajaj | Casava plants from meristem cultures freeze-preserved for three years | |
| WO2021238039A1 (en) | Rapid vitrification cryopreservation and recovery method of strongylocentrotus intermedius embryo | |
| CN111869656A (en) | Ultralow-temperature cryopreservation method for thamnaconus modestus sperms | |
| CN106614123B (en) | Method for inducing number-reducing gynogenesis of platichthys stellatus pallas by pressure shock | |
| CN108552161A (en) | A kind of the Ultra-cryofreezing preservation liquid and its Cryopreservation methods and applications of loach sperm | |
| CN110036950B (en) | Artificial crossbreeding method for mariculture of rainbow trout | |
| CN100348096C (en) | Vitrifiation freezing preservation method for genine porgy embryo particles | |
| CN112075415A (en) | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms | |
| CN113558044A (en) | Erythroculter ilishaeformis sperm cryopreservation technology | |
| Sakai | Cryopreservation of apical meristems. | |
| CN110999898A (en) | Method for cryopreservation of procypris merus sperm |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210618 |
|
| RJ01 | Rejection of invention patent application after publication |