CN112075415B - Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms - Google Patents
Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms Download PDFInfo
- Publication number
- CN112075415B CN112075415B CN202011022462.5A CN202011022462A CN112075415B CN 112075415 B CN112075415 B CN 112075415B CN 202011022462 A CN202011022462 A CN 202011022462A CN 112075415 B CN112075415 B CN 112075415B
- Authority
- CN
- China
- Prior art keywords
- sperm
- stichopus japonicus
- semen
- japonicus
- cooling
- 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.)
- Active
Links
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
-
- 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)
- Zoology (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Physiology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of ultralow-temperature cryopreservation of sea cucumber germplasm resources, in particular to a cryoprotectant and activator formula and an apostichopus japonicus sperm ultralow-temperature cryopreservation method based on a program cooling instrument and CASA. The freezing protection solution consists of diluent, antifreeze and additive; wherein the diluent is natural seawater, the antifreeze agent is one or more of aprotic polar solvents, and the additive is glucose. The method has large freezing storage amount (more than 200mL of fresh stichopus japonicus semen can be frozen in one batch), can precisely control the cooling process, has good application effect after thawing, and can solve the problems of asynchronous maturation of male and female sea cucumbers in north and south and the germplasm degradation of a series of stichopus japonicus in actual production.
Description
Technical Field
The invention relates to the technical field of ultralow-temperature cryopreservation of sea cucumber germplasm resources, in particular to a cryoprotectant and activator formula and an apostichopus japonicus sperm ultralow-temperature cryopreservation method based on a program cooling instrument and CASA.
Background
Apostichopus japonicus (Apostichopus japonicus), also known as Apostichopus japonicus, belongs to Echinodermata (Echinodermata), Holothuroidea (Holothuroidea), teno (Aspidochirodia), Apostichopaceae (Stichopodidae), Apostichopus japonicus (Apostichopus), naturally inhabits in the north of the Western pacific ocean, including the Russian far east coastal region, Japan and Korean coastal regions, the China yellow sea Bohai sea area, and the like, and is the most common higher invertebrate marine. Stichopus japonicus is a tonic food with homology of medicine and food, has the highest nutritional quality and economic value in more than 20 edible Stichopus japonicus distributed in the sea area of China, and has widely accepted health care and medical values since ancient times. With the market demand and the annual expansion of the stichopus japonicus, the excessive harvesting of the wild stichopus japonicus is getting more serious, so that the resource quantity and the germplasm quality of the wild stichopus japonicus are reduced sharply, and the stichopus japonicus is evaluated as Endangered (EN) Species by the Red List of endangered Species (IUCN Red List of threaded specificities or IUCN Red List) of the world natural protection alliance; the sea cucumber cultivation also has a series of germplasm degradation problems of frequent disease occurrence, low cultivation survival rate, slow growth speed and the like, the stichopus japonicus disease frequently occurs, about 30 hundred million yuan of economic loss is caused every year, the stichopus japonicus industry is seriously injured, and the method becomes an important bottleneck for restricting the healthy development of stichopus japonicus cultivation. The offshore environment influences the survival rate, quality and yield of the cultured sea cucumbers, and the sea cucumber culture itself influences the sea environment, so that the environmental pressure is increasingly severe, and even the sea cucumbers can not normally lay eggs, discharge sperms or have insufficient sperm-egg activity, so that the serious consequence of the incapability of breeding is caused. For example, in summer of 2018, seaside has been abused at high temperature, the lasting high-temperature weather is earlier than that of the past year and lasts for a long time, the cofferdam cultured sea cucumbers in Liaoning places, Shandong places and the like die greatly under the influence of high-temperature and high-humidity weather, and economic and germplasm resources are seriously lost. At present, researchers mostly adopt a south-north sea cucumber hybridization method with large geographical isolation to improve the quality of stichopus japonicus germplasm. However, the south-north male and female sea cucumbers are not matured synchronously, so that ripening and cleanup inducing measures are needed in the sea cucumber hybridization process, the operation is complex, the cost is high, and the problems that the hybridization cannot be realized due to ripening and cleanup inducing failure can also occur. Therefore, the problem of preservation of high-quality germplasm resources of stichopus japonicus is urgently to be solved.
In the current published papers or patents about ultra-low temperature cryopreservation methods for stichopus japonicus sperms, a two-step cooling mode (the height of a sample from the liquid level of liquid nitrogen is controlled to control the cooling rate) and a microscope are adopted to visually observe the sperm motility, the two modes have the influence of human subjective factors during operation, and the accuracy of results is greatly influenced by a cooling device and a counting device. A paper published by Shao et al (Shao M Y, Zhang Z F, Yu L, et al. Cryopression of sea cucumber Apostichopus japonicus (Selenka) sperm [ J ] 2006]Aquaculture Research,2006,37(14):1450-K,et al.Motility and fertility of cryopreserved spermatozoa of the Japanese sea cucumber Apostichopus japonicus[J]Aquaculture Research, 2018; 1-10.) diluting Stichopus japonicus sperm with preservation solution, and freezing and storing, wherein the preservation solution is artificial seawater (423.00mM NaCl, 9.00mM KCl, 9.27mM CaCl)2、22.94mM MgCl2、22.50mM MgSO4) 10mM Hepes buffer and 15% -20% dimethyl sulfoxide. The Hepes buffer solution is high in price and can generate hydrogen peroxide with certain biological toxicity when exposed to visible light; shao et al, with an operation time of 1 hour, have a large loss of sperm motility, and after thawing, the sperm life is only 1200s, which is difficult to apply to production; yuta Mizuno et al and 2019 (Heiyan, Zhan Yao Yao, Zhao Tan Jun et al, sea cucumber sperm cryopreservation method [ P)]CN110326610A,2019-10-15.) that the survival rate of all treated sperms after thawing is less than 15%, and the sperm motility rate, especially the ratio of sperm capable of moving forward, has a positive correlation with the fertilization rate and the hatching rate, and the higher the sperm motility after thawing is more beneficial to the long-term preservation and practical production and application of the sperms. However, the flow of the patent documents is not clear enough, and is greatly influenced by subjective factors of operators and a freezing device, and the methods in the patent documents are applied, so that the apostichopus japonicus sperm is not viable after being thawed.
Disclosure of Invention
The invention aims to solve the problems of asynchronous maturation of male and female sea cucumbers in south and north and the problem of germplasm degradation of a series of stichopus japonicus in actual production in the prior art, and provides a formula of an apostichopus japonicus sperm ultralow-temperature cryopreservation liquid, a formula of an activator and an apostichopus japonicus sperm ultralow-temperature cryopreservation method based on a program cooling instrument and CASA.
In order to achieve the purpose, the invention adopts the technical scheme that:
a freezing protection solution consists of diluent, antifreeze and additive; wherein the diluent is natural seawater, the antifreeze agent is one or more of aprotic polar solvents, and the additive is glucose.
The compound is dimethyl sulfoxide (DMSO) and/or Dimethylacetamide (DMA).
The freezing protection solution is natural seawater, dimethyl sulfoxide (DMSO) and glucose, wherein the volume mass ratio of the freezing protection solution to the glucose is 85 mL-90 mL: 10 mL-15 mL: 1.98 g.
The freezing protection solution is natural seawater and Dimethylacetamide (DMA), and the volume ratio is 80 mL: 20 mL.
The natural seawater is filtered by a 0.45-micron filter membrane; dimethyl sulfoxide (DMSO) is dimethyl sulfoxide with the purity of more than or equal to 99.97 percent, dimethyl acetamide (DMA) is dimethyl acetamide with the purity of more than or equal to 99.97 percent, and the DMSO and the DMA both belong to aprotic high-polarity solvents.
An ultra-low temperature cryopreservation method for stichopus japonicus sperm by using the cryoprotectant solution,
(1) semen obtaining: collecting the sperm of Stichopus japonicus in reproduction period;
(2) mixing the semen with the frozen preservation solution: fully mixing fresh stichopus japonicus semen and a freezing protection solution according to the volume ratio of 1: 5-1: 7, and placing the mixture into a container;
(3) and (3) programmed cooling: mixing, placing in a programmed cooling instrument, operating a cooling program, wherein the cooling program is to balance for 5min at 0 ℃, cool to-80 ℃ at a cooling rate of 10-15 ℃/min, balance for 5min at-80 ℃, cool to-180 ℃ at a cooling rate of 20 ℃/min, balance for 5min, and take out, thus realizing ultralow temperature cryopreservation of the apostichopus japonicus sperms.
And putting the sperm container subjected to the temperature reduction procedure into liquid nitrogen for long-term storage.
Unfreezing the frozen and preserved stichopus japonicus sperm in a water bath at 25 ℃, slightly shaking to ensure that the temperature is uniform, immediately taking out the stichopus japonicus sperm (about 120S) when only a small amount of solid remains, and continuously shaking in the air to completely melt the stichopus japonicus sperm; sucking frozen essence, adding into activator, mixing to make the dilution speed of original essence of Apostichopus japonicus up to 600 times, and the formula of activator is filtered natural seawater containing 5wt% fetal calf serum.
And (3) diluting the collected sperms by 600 times through an activator, detecting the sperms with the sperm collection activity of more than 90% for standby application.
The invention has the advantages that:
the cryopreservation method is based on two instruments, namely a program cooling instrument (Kasu Micro-Digitcool) and a CASA (Milang ML-608JZ), can accurately control the cooling rate and quantitatively analyze the sperm motility, and can rapidly cryopreserve the sperm in large batches (more than 200mL of fresh sperm can be cryopreserved in one batch); the operation time is short (the operation is completed within 20min after the sperm is dissected out), and the problem of great sperm motility loss is avoided; the used preservation solution has extremely low cost, safety, no toxicity, convenient preparation and good effect; after the sperm activating agent and the sperms are mixed, the sperms can be immediately activated and show high activity, and the high activity can be kept for more than 3 hours; after the frozen sperms are thawed and activated, the sperm motility rate is up to 60.07 +/-5.88%, the rapid sperm movement rate is up to 43.39 +/-5.88%, and the sperm curvilinear movement speed is up to 103.51 +/-15.46 mu m/s; the method provides convenience for the protection of stichopus japonicus germplasm resources, the large-scale production of seedlings and the genetic breeding research, has objective and credible data results, and is favorable for further detailed research on the ultra-low temperature cryopreservation method of stichopus japonicus sperms and the manufacturing of a freezing damage machine thereof. The concept of adopting an aprotic polar solvent in the refrigerating fluid is beneficial to developing and selecting a new antifreeze agent. The method has large freezing quantity (more than 200mL of fresh stichopus japonicus semen can be frozen in one batch), can precisely control the cooling process, has good application effect after thawing, and can solve the problems of asynchronous maturation of male and female sea cucumbers in north and south and the germplasm degradation of a series of stichopus japonicus in actual production.
The specific implementation mode is as follows:
the following examples are presented to further illustrate embodiments of the present invention, and it should be understood that the embodiments described herein are for purposes of illustration and explanation only and are not intended to limit the invention.
Aiming at the problems of low recovery rate, poor movement capability and the like in the existing ultra-low temperature cryopreservation of the apostichopus japonicus sperms, the invention obtains two cryopreservation liquids (including a sperm motility activating liquid) suitable for ultra-low temperature cryopreservation of the apostichopus japonicus sperms by carrying out systematic research on related factors of the apostichopus japonicus sperms cryopreservation. The preparation method of the preservation solution and the activating solution is simple, convenient to store and good in effect; the cryopreservation method has the advantages of long cryopreservation operation time, suitability for large-batch application, convenience for protection of stichopus japonicus germplasm resources, large-scale production of seedlings and genetic breeding research, and contribution to further detailed research on the cryopreservation method for the sperms of the stichopus japonicus and the manufacturing of a freezing damage machine of the sperms of the stichopus japonicus.
Example 1
a. Preparing a frozen preservation solution: preparing fresh ultra-low temperature freezing protection solution according to the existing preparation principle, wherein the formulation of the ultra-low temperature freezing protection solution for the apostichopus japonicus sperm is (1)85mL of natural seawater filtered by a 0.45-micrometer filter membrane, 15mL of dimethyl sulfoxide (DMSO) with the purity of more than or equal to 99.97 percent and 1.98g of glucose; formulation (2)80mL of natural seawater filtered through a 0.45 μm filter membrane, 20mL of Dimethylacetamide (DMA) having a purity of not less than 99.97%. And pre-cooling the prepared freezing protection liquid in a constant temperature refrigerator at 4 ℃ until the prepared freezing protection liquid is used.
b. Semen obtaining: collecting Stichopus japonicus in reproduction stage, and wiping off Stichopus japonicus body after it has drained water from body cavity (to avoid seawater contacting sperm to activate it). Dissecting sea cucumber from abdomen with sterile scalpel to avoid the gonad from being scratched, picking out complete gonad with forceps, sucking out the surface body fluid of gonad with absorbent paper, transferring to sterile culture dish, cutting with scissors, filtering with 300 mesh silk into sterile 50ml centrifuge tube, and placing in 4 deg.C constant temperature incubator for use. Meanwhile, the motility of fresh sperms is detected, and only the sperms with the motility more than 90 percent can be used for the freezing experiment. The sperm motility test comprises mixing 1198 μ l activator (filtered natural seawater containing 5wt% fetal calf serum) and 2 μ l fresh sperm, diluting by 600 times, activating, sucking 10 μ l mixed solution, adding into sperm counting plate, and testing by CASA.
c. Mixing the semen with the frozen preservation solution: the fresh stichopus japonicus semen and the freezing protection solution are fully mixed according to a certain volume ratio and then are subpackaged in a 2ml freezing tube, and the freezing tube is placed in a freezing tube box and placed in a heat preservation box with ice blocks in the whole process. The mixing volume ratio of the semen and the frozen preservation solution in the frozen protection solution formulas (1) and (2) is 1: 7. Taking 480 ul of total volume as an example, 420 ul of frozen protective solution and 60 ul of fresh stichopus japonicus semen are contained.
d. And (3) programmed cooling: placing the cryopreservation pipe frame for the cryopreservation pipe into a programmed cooling instrument, operating a cooling program, wherein the cooling program is to balance for 5min at 0 ℃, cool to-80 ℃ at a cooling rate of 15 ℃/min, balance for 5min at-80 ℃, cool to-180 ℃ at a cooling rate of 20 ℃/min, balance for 5min, take out, and put into liquid nitrogen (-196 ℃) for medium-long term storage;
e. unfreezing: taking out the freezing tube in the step d from the liquid nitrogen, unfreezing the freezing tube in a water bath at 25 ℃, slightly shaking to ensure that the temperature is uniform, immediately taking out the freezing tube when only a small amount of solid remains (about 120S), and continuously shaking in the air to ensure that the freezing tube is completely melted;
f. activation and sperm motility analysis: sucking frozen semen and adding into activator to make the total dilution multiple of fresh Apostichopus japonicus selenka semen reach 600 times, so as to keep 70-120 sperms in each visual field of CASA, if the volume ratio of the semen to the frozen preservation solution is 1:7, diluting 75 times, if 740 μ l of activator and 10 μ l of thawed frozen semen are mixed uniformly; 10 μ l of activated frozen sperm was aspirated, added to a sperm counting plate, and sperm motility analysis was performed using CASA.
And (3) displaying a statistical result: the sperm motility rate of the stichopus japonicus after being stored by the protective solution formula (1) and thawed and activated can reach 60.07 +/-5.88%, the sperm rapid movement rate can reach 43.39 +/-5.88%, and the sperm curvilinear movement speed can reach 103.51 +/-15.46 mu m/s; the sperm motility rate of the stichopus japonicus after being thawed and activated by the sperm stored by the protective solution formula (2) reaches 45.11 +/-5.22%, the sperm rapid movement rate reaches 26.68 +/-1.34%, and the sperm curvilinear movement speed reaches 136.54 +/-15.98 mu m/s.
Comparative example 1
a. Preparing a frozen preservation solution: according to the existing preparation principle, a fresh ultra-low temperature freezing protective solution is prepared, in the embodiment, the formulation of the ultra-low temperature freezing protective solution for the apostichopus japonicus sperms is 85mL of natural seawater filtered by a 0.45-micrometer filter membrane, and 15mL of dimethyl sulfoxide (DMSO) with the purity of more than or equal to 99.97 percent. And pre-cooling the prepared freezing protection liquid in a constant temperature refrigerator at 4 ℃ until the prepared freezing protection liquid is used.
b. Semen obtaining: same as example 1
c. Mixing the semen with the frozen preservation solution: fully mixing fresh stichopus japonicus semen and the freezing protection solution according to the volume ratio of 1:7, subpackaging in a 2ml freezing tube, and in the whole process, placing the freezing tube in a freezing tube box and placing in a heat preservation box with ice blocks.
d. And (3) programmed cooling: the same as example 1;
e. unfreezing: the same as example 1;
f. activation and sperm motility analysis: the same as example 1;
and (3) displaying a statistical result: in the embodiment, the sperm motility rate of the apostichopus japonicus after the sperm is thawed and activated is 46.29 +/-7.55%, the rapid sperm movement rate is 28 +/-2.44%, and the sperm curvilinear movement speed is 102.37 +/-6.62 mu m/s.
Comparative example 2
a. Preparing a frozen preservation solution: according to the existing preparation principle, a fresh ultra-low temperature freezing protective solution is prepared, and the formulation of the ultra-low temperature freezing protective solution for the apostichopus japonicus sperms in the embodiment is 80mL of natural seawater filtered by a 0.45-micron filter membrane, and 20mL of Propylene Glycol (PG) with the purity of more than or equal to 99.97 percent. And pre-cooling the prepared freezing protection liquid in a constant temperature refrigerator at 4 ℃ until the prepared freezing protection liquid is used.
b. Semen obtaining: same as example 1
c. Mixing the semen with the frozen preservation solution: same as example 1
d. And (3) programmed cooling: placing the cryopreservation pipe frame for the cryopreservation pipe into a programmed cooling instrument, operating a cooling program, wherein the cooling program is to balance for 5min at 0 ℃, cool to-80 ℃ at a cooling rate of 15 ℃/min, balance for 5min at-80 ℃, cool to-180 ℃ at a cooling rate of 20 ℃/min, balance for 5min, take out, and put into liquid nitrogen (-196 ℃) for medium-long term storage;
e. unfreezing: the same as example 1;
f. activation and sperm motility analysis: the same as example 1;
and (3) displaying a statistical result: in the embodiment, the sperm motility rate of the apostichopus japonicus after the sperm is thawed and activated is 7.84 +/-0.61%, the rapid sperm movement rate is 4.79 +/-0.96%, and the sperm curvilinear movement speed is 48.45 +/-12.94 mu m/s.
Comparative example 3
a. Preparing a frozen preservation solution: according to the existing preparation principle, a fresh ultra-low temperature freezing protective solution is prepared, in the embodiment, the formulation of the ultra-low temperature freezing protective solution for the apostichopus japonicus sperms is 85mL of natural seawater filtered by a 0.45-micrometer filter membrane, and 15mL of dimethyl sulfoxide (DMSO) with the purity of more than or equal to 99.97 percent. Pre-cooling the prepared freezing protection solution in a constant temperature refrigerator at 4 ℃ until the solution is used;
b. semen obtaining: the same as example 1;
c. mixing the semen with the frozen preservation solution: the same as example 1;
d. and (3) programmed cooling: placing the cryopreservation pipe frame for the cryopreservation pipe into a programmed cooling instrument, operating a cooling program, wherein the cooling program is to balance for 5min at 0 ℃, cool to-80 ℃ at a cooling rate of 25 ℃/min, balance for 5min at-80 ℃, cool to-180 ℃ at a cooling rate of 20 ℃/min, balance for 5min, take out, and put into liquid nitrogen (-196 ℃) for medium-long term storage;
e. unfreezing: the same as example 1;
f. activation and sperm motility analysis: the same as example 1;
and (3) displaying a statistical result: in the embodiment, the sperm motility rate of the apostichopus japonicus after the sperm is thawed and activated is 12.97 +/-0.46%, the rapid sperm movement rate is 7.83 +/-0.53%, and the sperm curvilinear movement rate is 80.28 +/-9.70 mu m/s.
Comparative example 4
a. Preparing a frozen preservation solution: according to the existing preparation principle, a fresh ultra-low temperature freezing protective solution is prepared, in the embodiment, the formulation of the ultra-low temperature freezing protective solution for the apostichopus japonicus sperms is 85mL of natural seawater filtered by a 0.45-micrometer filter membrane, and 15mL of dimethyl sulfoxide (DMSO) with the purity of more than or equal to 99.97 percent. Pre-cooling the prepared freezing protection solution in a constant temperature refrigerator at 4 ℃ until the solution is used;
b. semen obtaining: the same as example 1;
c. mixing the semen with the frozen preservation solution: fully mixing fresh stichopus japonicus semen and the freezing protection solution according to the volume ratio of 1:19, subpackaging in a 2ml freezing tube, and in the whole process, placing the freezing tube in a freezing tube box and placing in a heat preservation box with ice blocks. Taking 480 μ l of total volume as an example, 456 μ l of frozen protective solution and 24 μ l of fresh stichopus japonicus semen are contained;
d. and (3) programmed cooling: the same as example 1;
e. unfreezing: the same as example 1;
f. activation and sperm motility analysis: sucking frozen semen and adding into activator to make the total dilution multiple of fresh Stichopus japonicus reach 600 times, so as to keep 70-120 sperms in each visual field of CASA, if the volume ratio of semen to frozen preservation solution is 1:19, diluting 30 times, if 290 μ l of activating solution and 10 μ l of thawed frozen semen are mixed uniformly; 10 μ l of activated frozen sperm was aspirated, added to a sperm counting plate, and sperm motility analysis was performed using CASA.
And (3) displaying a statistical result: in the embodiment, the sperm motility rate of the apostichopus japonicus after the sperm is thawed and activated is 13.18 +/-0.74%, the rapid sperm movement rate is 9.24 +/-0.52%, and the curvilinear sperm movement rate is 101.34 +/-2.15 mu m/s.
According to the specific refrigerating fluid and the ultralow-temperature freezing treatment of the apostichopus japonicus sperm under specific conditions, the activated sperm activity can reach 60.07 +/-5.88%, the sperm rapid movement ratio can reach 43.39 +/-5.88%, the sperm curvilinear movement speed can reach 103.51 +/-15.46 mu m/s, the aprotic high-polarity solvent in the refrigerating fluid can rapidly permeate into the cells and is combined with water and electrolyte to generate certain molar concentration in the cells, the concentration of the electrolyte solution of the unfrozen solution inside and outside the cells is reduced, the freezing point is lowered, the formation of ice crystals is reduced, and meanwhile, the phenomenon that the water in the cells excessively seeps to cause cell shrinkage to cause deposition damage is avoided; fully mixing fresh stichopus japonicus semen and a freezing protection solution according to the volume ratio of 1: 5-1: 7, and then cooling to-80 ℃ at the optimal cooling rate (10-15 ℃/min), so that the fresh stichopus japonicus semen rapidly crosses a dangerous area (0-60 ℃) and simultaneously avoids more ice crystal damage to sperms caused by the excessively high cooling rate, and further, the apostichopus japonicus sperms are preserved for a long time (liquid nitrogen-196 ℃); the specific activator is used for activating the sperm to enable the vitality of the sperm to reach the maximum value instantly, and the high vitality of the sperm is kept for more than 3 hours, so that the sperm vitality can be detected more efficiently and the practical application is facilitated; even under the conditions that the same refrigerating fluid is adopted, but under different mixing ratios, different cooling rates or other types of antifreeze agents are adopted, and the like, the stichopus japonicus sperms are subjected to more freezing damage in the ultralow temperature freezing process due to the fact that the conditions are not suitable, and the corresponding effect cannot be achieved after the stichopus japonicus sperms are activated after being frozen, and therefore the technical effect provided by the scheme provided by the embodiment of the invention is unexpected.
Claims (6)
1. A cryoprotectant solution, characterized by:
the freezing protection solution consists of natural seawater, dimethyl sulfoxide (DMSO) and glucose, wherein the volume mass ratio of each is 85 mL-90 mL: 10 mL-15 mL: 1.98 g;
the freezing protection solution consists of natural seawater and Dimethylacetamide (DMA), and the volume ratio is 80 mL: 20 mL;
the freezing protection solution is used for freezing and storing fresh stichopus japonicus semen, and the volume ratio of the freezing protection solution to the fresh stichopus japonicus semen is 1: 7.
2. A cryoprotectant solution according to claim 1, wherein: the natural seawater is filtered by a 0.45-micron filter membrane; dimethyl sulfoxide (DMSO) is dimethyl sulfoxide with the purity of more than or equal to 99.97 percent, dimethyl acetamide (DMA) is dimethyl acetamide with the purity of more than or equal to 99.97 percent, and the DMSO and the DMA both belong to aprotic high-polarity solvents.
3. A method for cryopreservation of Stichopus japonicus sperm using the cryoprotectant solution of claim 1, comprising the steps of
(1) Semen obtaining: collecting the sperm of Stichopus japonicus in reproduction period;
(2) mixing the semen with the frozen preservation solution: fully mixing fresh stichopus japonicus semen and the freezing protection solution according to the volume ratio of 1:7, and placing the mixture into a container;
(3) and (3) programmed cooling: mixing, placing in a programmed cooling instrument, operating a cooling program, wherein the cooling program is to balance for 5min at 0 ℃, cool to-80 ℃ at a cooling rate of 15 ℃/min, balance for 5min at-80 ℃, cool to-180 ℃ at a cooling rate of 20 ℃/min, balance for 5min, and take out, thereby realizing ultralow temperature cryopreservation of the stichopus japonicus sperms.
4. The method of claim 3, wherein: and putting the sperm container subjected to programmed cooling into liquid nitrogen for long-term storage.
5. The method of claim 3, wherein: unfreezing the frozen and preserved stichopus japonicus sperm in a water bath at 25 ℃, slightly shaking to ensure that the temperature is uniform, immediately taking out the stichopus japonicus sperm when only a small amount of solid remains in the stichopus japonicus sperm about 120 seconds, and continuously shaking in the air to completely melt the stichopus japonicus sperm; sucking frozen essence, adding into activator, mixing to make the dilution speed of original essence of Apostichopus japonicus up to 600 times, and the formula of activator is filtered natural seawater containing 5wt% fetal calf serum.
6. The method of claim 3, wherein: and (3) diluting the collected sperms by 600 times through an activator, detecting the sperms with the sperm collection activity of more than 90% for standby application.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011022462.5A CN112075415B (en) | 2020-09-25 | 2020-09-25 | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms |
NL2028946A NL2028946B1 (en) | 2020-09-25 | 2021-08-10 | Cryoprotective solution and ultra-low temperature cryopreservation method of stichopus japonicus sperm |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011022462.5A CN112075415B (en) | 2020-09-25 | 2020-09-25 | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112075415A CN112075415A (en) | 2020-12-15 |
CN112075415B true CN112075415B (en) | 2021-08-31 |
Family
ID=73738855
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011022462.5A Active CN112075415B (en) | 2020-09-25 | 2020-09-25 | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN112075415B (en) |
NL (1) | NL2028946B1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116034992B (en) * | 2023-03-08 | 2023-06-27 | 中国科学院海洋研究所 | Low-temperature stichopus japonicus sperm preservation solution and application and stichopus japonicus sperm preservation method |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101326905A (en) * | 2008-08-01 | 2008-12-24 | 集美大学 | Method for preserving Porphyra haitanensis young seedling at low-temperature |
CN103348966A (en) * | 2013-05-31 | 2013-10-16 | 中国科学院海洋研究所 | Method for efficient ultralow temperature cryopreservation of turbot sperms |
CN104336004A (en) * | 2013-07-24 | 2015-02-11 | 中国科学院海洋研究所 | Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms |
CN105123580A (en) * | 2015-09-06 | 2015-12-09 | 莱州明波水产有限公司 | Industrialized hybrid fry breeding method for saladfish and lanceolatus |
CN105850814A (en) * | 2016-05-19 | 2016-08-17 | 莱州明波水产有限公司 | Industrialized hybrid fry breeding method for chromileptes altivelis and epinephelus lanceolatus |
CN106614522A (en) * | 2016-10-18 | 2017-05-10 | 淮海工学院 | Urechis unicinctus sperm cryopreservation liquid and preparation method thereof |
CN106857497A (en) * | 2015-12-11 | 2017-06-20 | 中国科学院海洋研究所 | A kind of seven methods preserved with grouper sperm super-low temperature |
CN110326610A (en) * | 2019-07-19 | 2019-10-15 | 大连海洋大学 | Sea cucumber sperm cryopreservation method |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6689391B2 (en) * | 2001-03-30 | 2004-02-10 | Council Of Scientific & Industrial Research | Natural non-polar fluorescent dye from a non-bioluminescent marine invertebrate, compositions containing the said dye and its uses |
US20080085329A1 (en) * | 2003-10-22 | 2008-04-10 | Fred Hutchinson Cancer Research Center, Inc. | Methods, Compositions and Devices for Inducing Stasis in Cells, Tissues, Organs, and Organisms |
CA2605631A1 (en) * | 2005-04-20 | 2006-10-26 | Fred Hutchinson Cancer Research Center | Methods, compositions and articles of manufacture for enhancing survivability of cells, tissues, organs, and organisms |
CN104145944B (en) * | 2014-08-25 | 2016-05-11 | 山东省海洋生物研究院 | A kind of stalwart blood clam sperm super-low temperature freezing is preserved and Activiation method |
CN109329272B (en) * | 2018-11-23 | 2020-03-03 | 北京太东生物科技有限公司 | Sperm cryopreservation liquid and preparation method and application thereof |
-
2020
- 2020-09-25 CN CN202011022462.5A patent/CN112075415B/en active Active
-
2021
- 2021-08-10 NL NL2028946A patent/NL2028946B1/en active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101326905A (en) * | 2008-08-01 | 2008-12-24 | 集美大学 | Method for preserving Porphyra haitanensis young seedling at low-temperature |
CN103348966A (en) * | 2013-05-31 | 2013-10-16 | 中国科学院海洋研究所 | Method for efficient ultralow temperature cryopreservation of turbot sperms |
CN104336004A (en) * | 2013-07-24 | 2015-02-11 | 中国科学院海洋研究所 | Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms |
CN105123580A (en) * | 2015-09-06 | 2015-12-09 | 莱州明波水产有限公司 | Industrialized hybrid fry breeding method for saladfish and lanceolatus |
CN106857497A (en) * | 2015-12-11 | 2017-06-20 | 中国科学院海洋研究所 | A kind of seven methods preserved with grouper sperm super-low temperature |
CN105850814A (en) * | 2016-05-19 | 2016-08-17 | 莱州明波水产有限公司 | Industrialized hybrid fry breeding method for chromileptes altivelis and epinephelus lanceolatus |
CN106614522A (en) * | 2016-10-18 | 2017-05-10 | 淮海工学院 | Urechis unicinctus sperm cryopreservation liquid and preparation method thereof |
CN110326610A (en) * | 2019-07-19 | 2019-10-15 | 大连海洋大学 | Sea cucumber sperm cryopreservation method |
Non-Patent Citations (2)
Title |
---|
Cryopreservation of sea cucumber Apostichopus japonicus (Selenka) sperm;Shao M Y等;《Aquaculture Research》;20061231;第37卷(第14期);第1450-1457页 * |
冷冻温度对牛精子复苏率及降温速率变化规律的研究;邓福金等;《中国牛业科学》;20101231(第5期);第27-29页 * |
Also Published As
Publication number | Publication date |
---|---|
NL2028946A (en) | 2022-05-24 |
CN112075415A (en) | 2020-12-15 |
NL2028946B1 (en) | 2022-06-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Withers | Cryopreservation of cultured plant cells and protoplasts | |
CN112741078B (en) | Hexagrammos otakii sperm productive cryopreservation method | |
CN110326610B (en) | Ultralow temperature cryopreservation method for sea cucumber sperms | |
CN108641999B (en) | Cryopreservation and recovery method of ovarian tissue | |
CN114903032A (en) | Preparation method of frozen semen of Dongfrui raw milk sheep | |
CN112075415B (en) | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms | |
US20230276790A1 (en) | Hypothermic preserving diluent, hypothermic preserving method of plectropomus leopardus semen and application thereof | |
CN113273567A (en) | Low-temperature preservation liquid for patinopecten yessoensis sperms and preservation and use method | |
CN104145944A (en) | Ultralow-temperature cryopreservation and activation method of sperm of scapharca broughtonii sckrenck | |
CN110100813B (en) | Sheep embryo vitrification cryopreservation fluid formula and freezing method | |
CN111869656A (en) | Ultralow-temperature cryopreservation method for thamnaconus modestus sperms | |
CN116076485A (en) | Ultra-low temperature freezing protection liquid for grape dental oyster sperm and ultra-low temperature freezing preservation method | |
CN112293407A (en) | Method for programmed cryopreservation of ovarian tissues | |
CN104336004A (en) | Method for collection and ultralow temperature refrigeration preservation of high-quality pacific oyster sperms | |
CN112931490B (en) | Ultralow temperature cryopreservation method for bee sperms | |
JP6983452B2 (en) | Ultra-low temperature cryopreservation method for sea cucumber sperm | |
CN112293411B (en) | Low-temperature preservation liquid and preservation method for sperms of echinococcus intermedius | |
CN100348096C (en) | Vitrifiation freezing preservation method for genine porgy embryo particles | |
CN111972397A (en) | Skin cold storage preservation solution and application thereof | |
CN110999898A (en) | Method for cryopreservation of procypris merus sperm | |
CN117898229B (en) | Artificial fertilization method for Chlamys nobilis | |
CN115067322B (en) | Method for preserving Selaginella schlegeli sperms in short term by high-activity non-freezing | |
CN116034992B (en) | Low-temperature stichopus japonicus sperm preservation solution and application and stichopus japonicus sperm preservation method | |
CN110521721B (en) | Method for freezing and storing grouper embryos by utilizing non-permeable antifreeze agent and open carrier | |
CN115074315B (en) | High-quality pig ear tissue sampling and long-term high-efficiency preservation method |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |