CN110999898A - Method for cryopreservation of procypris merus sperm - Google Patents
Method for cryopreservation of procypris merus sperm Download PDFInfo
- Publication number
- CN110999898A CN110999898A CN201911228597.4A CN201911228597A CN110999898A CN 110999898 A CN110999898 A CN 110999898A CN 201911228597 A CN201911228597 A CN 201911228597A CN 110999898 A CN110999898 A CN 110999898A
- Authority
- CN
- China
- Prior art keywords
- sperm
- semen
- solution
- liquid nitrogen
- procypris merus
- 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
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
- A01N1/0278—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)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a procypris merus sperm cryopreservation method which comprises the four steps of semen collection, semen dilution, sperm cryopreservation and sperm thawing, wherein the semen of the procypris merus is collected by adopting an artificial abdominal squeezing method, then the semen is mixed with a diluent and then is mixed with an anti-freezing solution to obtain a sperm preservation solution, and the diluent is any one of D-15 diluent, Hank's solution and Nigella flexneri solution for fish; the antifreeze is any one of methanol, dimethyl sulfoxide and glycerol; injecting the sperm preservation solution into a freezing tube, and immersing the tube in liquid nitrogen for long-term preservation; when the procypris merus sperm preservation solution is taken out from liquid nitrogen, the procypris merus sperm preservation solution is immediately placed in a water bath kettle at the temperature of 27 ℃, 37 ℃ or 47 ℃ for unfreezing, and the obtained procypris merus sperm is used for artificial insemination. The method can preserve the sperm of the procypris merus for a long time, and the sperm survival rate is high after thawing recovery.
Description
Technical Field
The invention belongs to the technical field of biological low-temperature cryopreservation, and particularly relates to a procypris merus sperm cryopreservation method.
Background
The procypris merus belongs to family Cyprinaceae and is a small warm-water fish, also called procypris merus, procypris merus and procypris, is thick in body, slightly purple (dark brown) in the whole body, black in back and bright in color, is originally produced in Guangxi Guilin, belongs to native fishes in China, and is a breeding variety with local characteristics. The procypris merus has fine meat quality, less thorns and more meat, soft bones, no fishy smell, delicious taste and high protein content, and the procypris merus is polycultured in a rice field, has poor feeding performance, fast growth, strong reproductive capacity and short culture period and has high nutritional value and economic value.
With the increasing of the breeding scale of the procypris merus year by year, the demand on the procypris merus seedlings is gradually increased, and the quality requirement on the procypris merus seedlings is also increased year by year. The artificial propagation can ensure that the procypris merus lays eggs in a centralized way, improve the fertilization rate, the hatching rate and the survival rate of fish fries, and realize planned production. The breeding of the procypris merus has seasonality, and the sperm concentration and the sperm activity of male individuals of the procypris merus are low and reduced in non-breeding seasons, so that the sperm of the procypris merus needs to be collected and stored for a long time in the breeding seasons, the whole-year breeding and production needs of the procypris merus are facilitated, the artificial insemination efficiency and the seedling survival rate of the procypris merus in the non-breeding seasons are improved, and at present, few methods capable of storing the sperm of the procypris merus for a long time and recovering the sperm are available.
Disclosure of Invention
Aiming at the defects, the invention discloses a procypris merus sperm cryopreservation method, which can be used for preserving procypris merus sperm for a long time and has high sperm survival rate after thawing recovery.
The invention is realized by adopting the following technical scheme:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 10% or 20% or 30% of antifreeze agent, and the balance of diluent; the diluent is any one of D-15 diluent, Hank's solution and Nilerian solution for fish; the antifreeze is any one of methanol, dimethyl sulfoxide and glycerol;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube at a position 4cm above the surface of liquid nitrogen for staying for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; or placing the freezing tube at 4 deg.C, balancing for 30min, placing the freezing tube 8cm above the liquid nitrogen surface, standing for 5min, and soaking the freezing tube in liquid nitrogen for long-term storage; or balancing the freezing tube at 4 deg.C for 30min, standing for 5min at 8cm above the liquid nitrogen surface, standing for 5min at 4cm above the liquid nitrogen surface, and soaking the freezing tube in liquid nitrogen for long-term storage;
(4) and (3) thawing the sperms: taking out the frozen tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 27 ℃, 37 ℃ or 47 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
Further, the D-15 diluent in the step (2) is obtained by adding 0.8g of NaCl, 0.05g of KCl and 1.5g of glucose into each 100ml of pure water and mixing; the Hank's solution is prepared by adding 0.801g NaCl, 0.04g KCl and 0.014g CaCl into 100ml pure water2、 0.035g NaHCO3、0.006g KH2PO4And 0.034 g glucose; the fish ringer's solution is prepared by adding 0.78g NaCl and 0.021g CaCl into 100ml pure water2、0.02g KCl、0.2g NaHCO3Mixing the obtained mixture.
Further, the temperature at the position 4cm above the surface of the liquid nitrogen in the step (3) is-92 to-88 ℃; the temperature 8cm above the liquid nitrogen surface is-22 to-18 ℃.
Further, the anti-freezing solution in the step (2) is stored at the temperature of 4 ℃ for later use.
Further, 10 microliters of the collected semen is mixed with equal volume of potassium chloride with the mass fraction of 0.35% in the step (1), the sperm movement condition is rapidly observed under a microscope, and the semen with the sperm motility rate of more than 90% is selected for dilution and preservation. The semen with high sperm activity is selected for dilution and preservation, which is beneficial to the long-term preservation of procypris merus sperm and improves the survival rate of the thawed and revived sperm.
Compared with the prior art, the technical scheme has the following beneficial effects:
1. according to the sperm characteristics of procypris merus, the diluent and the anti-freezing solution are reasonably prepared to dilute the semen of procypris merus, and then the semen is cooled step by step, so that the influence on the sperm preservation effect and the survival rate of the thawed sperm caused by the damage of ice crystals and oxidation to the sperm is prevented.
2. In the process of cryopreservation, three diluents, three anti-freezing liquids, three cooling methods and three resuscitation temperatures are set, and a single variable is strictly controlled to screen freezing conditions, so that the combination of the obtained cryopreservation conditions has important production reference significance.
3. Before cooling, the diluent and the antifreeze protective agent are mixed to prepare the antifreeze mixed liquid, and the antifreeze mixed liquid is stored in a refrigerator at 4 ℃ for later use, so that the operation is simple, convenient and quick, and the time is saved.
4. The method disclosed by the invention is simple to operate, good in cryopreservation effect and low in cost, can be used for preserving the sperms of the procypris merus for a long time, realizing the permanent preservation of germplasm resources of the procypris merus, and can be used for selecting and preserving a large amount of high-quality semen in the breeding season of the procypris merus for the annual artificial breeding production of the procypris merus.
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. The specific experimental conditions and methods not indicated in the following examples are generally conventional means well known to those skilled in the art.
Example 1:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube; mixing 10 microliters of collected semen with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the movement condition of the semen under a microscope, and selecting the semen with high sperm activity for dilution and storage;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 20% of antifreeze agent, and the balance of diluent; the diluent is D-15 diluent; the antifreeze agent is dimethyl sulfoxide; the D-15 diluent is obtained by adding 0.8g of NaCl, 0.05g of KCl and 1.5g of glucose into each 100ml of pure water and mixing; the anti-freezing liquid is stored at the temperature of 4 ℃ for later use;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube at a position 4cm above the surface of liquid nitrogen for staying for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; the temperature 4cm above the liquid nitrogen surface is-90 ℃;
(4) and (3) thawing the sperms: taking out the cryopreservation tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 37 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
The procypris merus sperm is frozen and preserved according to the method of the embodiment, 10 microliter of unfrozen semen is sucked and mixed with equal volume of potassium chloride with the mass fraction of 0.35%, the sperm movement condition is rapidly observed under a microscope, after activation, the frozen sperm survival rate is 79.43% +/-1.01%, the average linear velocity is 8.35 +/-4.92 microns/second, the average curve velocity is 25.97 +/-9.72 microns/second, and the average path velocity is 22.46 +/-8.76 microns/second through microscope detection.
Example 2:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube; mixing 10 microliters of collected semen with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the movement condition of the semen under a microscope, and selecting the semen with high sperm activity for dilution and storage;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 20% of antifreeze agent, and the balance of diluent; the diluent is D-15 diluent; the antifreeze agent is glycerol; the D-15 diluent is obtained by adding 0.8g of NaCl, 0.05g of KCl and 1.5g of glucose into each 100ml of pure water and mixing; the anti-freezing liquid is stored at the temperature of 4 ℃ for later use;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube 8cm above the surface of liquid nitrogen for 5min, then placing the freezing tube 4cm above the surface of the liquid nitrogen for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; the temperature 4cm above the liquid nitrogen surface is-92 ℃; the temperature 8cm above the liquid nitrogen surface is-20 ℃;
(4) and (3) thawing the sperms: taking out the cryopreservation tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 47 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
The procypris merus sperm is frozen and preserved according to the method of the embodiment, 10 microliters of unfrozen semen is sucked and mixed with equal volume of potassium chloride with the mass fraction of 0.35%, and the sperm movement condition is rapidly observed under a microscope, after activation, the frozen sperm survival rate is 81.19% +/-2.98%, the average linear velocity is 11.42 +/-0.36 micrometers/second, the average curve velocity is 32.45 +/-2.75 micrometers/second, and the average path velocity is 28.25 +/-2.38 micrometers/second through microscope detection.
Example 3:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube; mixing 10 microliters of collected semen with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the movement condition of the semen under a microscope, and selecting the semen with high sperm activity for dilution and storage;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 10% of antifreeze agent, and the balance of diluent; the diluent is Hank's solution; the antifreeze agent is dimethyl sulfoxide; the Hank's solution is prepared by adding 0.801g NaCl, 0.04g KCl and 0.014g CaCl into 100ml pure water2、 0.035g NaHCO3、0.006g KH2PO4And 0.034 g glucose; the anti-freezing liquid is stored at the temperature of 4 ℃ for later use;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube at a position 4cm above the surface of liquid nitrogen for staying for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; the temperature at the position 4cm above the liquid nitrogen surface is-88 ℃;
(4) and (3) thawing the sperms: taking out the cryopreservation tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 27 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
The procypris merus sperm is frozen and preserved according to the method of the embodiment, 10 microliter of unfrozen semen is sucked and mixed with equal volume of potassium chloride with the mass fraction of 0.35%, the sperm movement condition is rapidly observed under a microscope, after activation, the frozen sperm survival rate is 77.29 +/-2.56%, the average linear velocity is 10.34 +/-0.88 microns/second, the average curve velocity is 28.32 +/-1.99 microns/second, and the average path velocity is 24.72 +/-1.62 microns/second through the microscope detection.
Example 4:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube; mixing 10 microliters of collected semen with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the movement condition of the semen under a microscope, and selecting the semen with high sperm activity for dilution and storage;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 10% of antifreeze agent, and the balance of diluent; the diluent is a wilsonian liquid for fish; the antifreeze agent is methanol; the fish ringer's solution is prepared by adding 0.78g NaCl and 0.021g CaCl into 100ml pure water2、0.02g KCl、0.2g NaHCO3Mixing to obtain the product; the anti-freezing solution is preserved at the temperature of 4 DEG CUsing;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube 8cm above the surface of liquid nitrogen for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; the temperature 8cm above the liquid nitrogen surface is-18 ℃;
(4) and (3) thawing the sperms: taking out the cryopreservation tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 37 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
The procypris merus sperm is frozen and preserved according to the method of the embodiment, 10 microliters of unfrozen semen is sucked and mixed with equal volume of potassium chloride with the mass fraction of 0.35%, and the sperm movement condition is rapidly observed under a microscope, after activation, the frozen sperm survival rate is 75.45% +/-2.03%, the average linear velocity is 10.83 +/-7.45 microns/second, the average curve velocity is 28.62 +/-8.99 microns/second, and the average path velocity is 25.07 +/-8.69 microns/second.
Example 5:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube; mixing 10 microliters of collected semen with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the movement condition of the semen under a microscope, and selecting the semen with high sperm activity for dilution and storage;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 30 percent of antifreeze agent, and the balance of diluentReleasing the liquid; the diluent is Hank's solution; the antifreeze agent is glycerol; the Hank's solution is prepared by adding 0.801g NaCl, 0.04g KCl and 0.014g CaCl into 100ml pure water2、 0.035g NaHCO3、0.006g KH2PO4And 0.034 g glucose; the anti-freezing liquid is stored at the temperature of 4 ℃ for later use;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube 8cm above the surface of liquid nitrogen for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; the temperature 8cm above the liquid nitrogen surface is-22 ℃;
(4) and (3) thawing the sperms: taking out the cryopreservation tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 27 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
The procypris merus sperm is frozen and preserved according to the method of the embodiment, 10 microliter of unfrozen semen is sucked and mixed with equal volume of potassium chloride with the mass fraction of 0.35%, the sperm movement condition is rapidly observed under a microscope, after activation, the frozen sperm activity is detected by the microscope to be 75.99 +/-2.79%, the average linear velocity is 4.29 +/-0.39 microns/second, the average curve velocity is 20.97 +/-2.26 microns/second, and the average path velocity is 17.63 +/-1.80 microns/second.
Comparative example 1:
a procypris merus sperm cryopreservation method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube; mixing 10 microliters of collected semen with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the movement condition of the semen under a microscope, and selecting the semen with high sperm activity for dilution and storage;
（2）semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:4 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 2:3 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 30% of antifreeze agent, and the balance of diluent; the diluent is a wilsonian liquid for fish; the antifreeze agent is methanol; the fish ringer's solution is prepared by adding 0.78g NaCl and 0.021g CaCl into 100ml pure water2、0.02g KCl、0.2g NaHCO3Mixing to obtain the product; the anti-freezing liquid is stored at the temperature of 4 ℃ for later use;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube above the surface of liquid nitrogen and staying at-16 ℃ for 5min, then placing the freezing tube above the surface of the liquid nitrogen and staying at-95 ℃ for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation;
(4) and (3) thawing the sperms: taking out the cryopreservation tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 50 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
The procypris merus sperm is frozen and preserved according to the method of the comparative example, then 10 microliter of unfrozen semen is sucked and mixed with equal volume of potassium chloride with the mass fraction of 0.35 percent, the sperm movement situation is rapidly observed under a microscope, after activation, the frozen sperm survival rate is 58.90 percent +/-1.08 percent, the average linear velocity is 5.77 +/-3.29 microns/second, the average curve velocity is 23.89 +/-4.50 microns/second, and the average path velocity is 20.27 +/-4.22 microns/second.
The procypris merus sperm preserved according to the methods in the embodiments 1 to 5 have the frozen sperm survival rate improved by at least 15 percent compared with the procypris merus sperm preserved according to the method in the comparative example 1, and the average path speed is also improved to a certain extent, which shows that the method has good frozen preservation effect and can preserve the procypris merus sperm for a long time.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (5)
1. A procypris merus sperm cryopreservation method is characterized in that: the method comprises the following steps:
(1) semen collection: in the breeding season of procypris merus, selecting a strong male parent fish with mature gonads, wiping water and mucus around the genital pore of the fish body by using a dry towel after anesthesia, slightly pressing the abdomen along the midline until the genital pore flows out milky semen, sucking the semen by using an injector to prevent urine, blood and water from mixing, and injecting the collected semen into a cryopreservation tube;
(2) semen dilution: mixing and diluting the semen collected in the step (1) and the diluent according to the volume ratio of 1:3 to obtain a solution A, and then mixing the solution A and the antifreeze according to the volume ratio of 1:1 to obtain a sperm preservation solution of procypris merus; the anti-freezing liquid consists of the following components in percentage by volume: 10% or 20% or 30% of antifreeze agent, and the balance of diluent; the diluent is any one of D-15 diluent, Hank's solution and Nilerian solution for fish; the antifreeze is any one of methanol, dimethyl sulfoxide and glycerol;
(3) and (3) sperm cryopreservation: injecting the sperm preservation solution obtained in the step (2) into a freezing tube, then balancing the freezing tube at 4 ℃ for 30min, then placing the freezing tube at a position 4cm above the surface of liquid nitrogen for staying for 5min, and then immersing the freezing tube into the liquid nitrogen for long-term preservation; or placing the freezing tube at 4 deg.C, balancing for 30min, placing the freezing tube 8cm above the liquid nitrogen surface, standing for 5min, and soaking the freezing tube in liquid nitrogen for long-term storage; or balancing the freezing tube at 4 deg.C for 30min, standing for 5min at 8cm above the liquid nitrogen surface, standing for 5min at 4cm above the liquid nitrogen surface, and soaking the freezing tube in liquid nitrogen for long-term storage;
(4) and (3) thawing the sperms: taking out the frozen tube filled with the sperm preservation solution from liquid nitrogen, then immediately placing the tube in a water bath kettle at the temperature of 27 ℃, 37 ℃ or 47 ℃ for thawing, and then placing the thawed sperm preservation solution at the temperature of 4 ℃ for later use.
2. The procypris merus sperm cryopreservation method as claimed in claim 1, wherein: the D-15 diluent in the step (2) is obtained by adding 0.8g of NaCl, 0.05g of KCl and 1.5g of glucose into each 100ml of pure water and mixing; the Hank's solution is prepared by adding 0.801g NaCl, 0.04g KCl and 0.014g CaCl into 100ml pure water2、 0.035gNaHCO3、0.006g KH2PO4And 0.034 g glucose; the fish ringer's solution is prepared by adding 0.78g NaCl and 0.021g CaCl into 100ml pure water2、0.02g KCl、0.2g NaHCO3Mixing the obtained mixture.
3. The procypris merus sperm cryopreservation method as claimed in claim 1, wherein: the temperature 4cm above the surface of the liquid nitrogen in the step (3) is-92 to-88 ℃; the temperature 8cm above the liquid nitrogen surface is-22 to-18 ℃.
4. The procypris merus sperm cryopreservation method as claimed in claim 1, wherein: and (3) storing the anti-freezing liquid at the temperature of 4 ℃ for later use in the step (2).
5. The procypris merus sperm cryopreservation method as claimed in claim 1, wherein: mixing 10 microliters of the collected semen in the step (1) with equal volume of potassium chloride with the mass fraction of 0.35%, rapidly observing the sperm movement condition under a microscope, and selecting semen with the sperm motility rate of more than 90% for dilution and preservation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911228597.4A CN110999898A (en) | 2019-12-04 | 2019-12-04 | Method for cryopreservation of procypris merus sperm |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911228597.4A CN110999898A (en) | 2019-12-04 | 2019-12-04 | Method for cryopreservation of procypris merus sperm |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110999898A true CN110999898A (en) | 2020-04-14 |
Family
ID=70115337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911228597.4A Pending CN110999898A (en) | 2019-12-04 | 2019-12-04 | Method for cryopreservation of procypris merus sperm |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110999898A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931488A (en) * | 2021-02-26 | 2021-06-11 | 内江师范学院 | Efficient ultralow-temperature cryopreservation method for Amyda sinensis (Wiegmann) sperms |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
| CN101884322A (en) * | 2010-07-20 | 2010-11-17 | 中国水产科学研究院黄海水产研究所 | Verasper moseri sperm cryopreservation method |
| CN104521943A (en) * | 2014-12-09 | 2015-04-22 | 南京师范大学 | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen |
-
2019
- 2019-12-04 CN CN201911228597.4A patent/CN110999898A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
| CN101884322A (en) * | 2010-07-20 | 2010-11-17 | 中国水产科学研究院黄海水产研究所 | Verasper moseri sperm cryopreservation method |
| CN104521943A (en) * | 2014-12-09 | 2015-04-22 | 南京师范大学 | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen |
Non-Patent Citations (1)
| Title |
|---|
| 周磊 等: "斑鳜精液超低温冷冻保存及其效果分析", 《中国水产科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931488A (en) * | 2021-02-26 | 2021-06-11 | 内江师范学院 | Efficient ultralow-temperature cryopreservation method for Amyda sinensis (Wiegmann) sperms |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110881460A (en) | Cryopreservation method for sperm of Oplophorus argus | |
| CN112741078B (en) | Hexagrammos otakii sperm productive cryopreservation method | |
| CN102077823B (en) | Method for cryopreservation of semen of siniperca scherzeri and artificial insemination | |
| CN104145944B (en) | A kind of stalwart blood clam sperm super-low temperature freezing is preserved and Activiation method | |
| CN103348966B (en) | Method for efficient ultralow temperature cryopreservation of turbot sperms | |
| CN106417251B (en) | High-concentration vitrification solution and ultra-low temperature cryopreservation method for grouper embryos | |
| 郭金泉 et al. | Cryopreservation of spermatozoa from rainbow trout, common carp, and marine puffer. | |
| CN110786321A (en) | Freeze preservation method for pseudosciaena crocea sperms | |
| CN105360109B (en) | A kind of Pelteobagrus fulvidraco sperm super-low temperature freezing store method | |
| CN114258911B (en) | Cryopreservation solution and cryopreservation method for sparus latus sperms | |
| CN109819976B (en) | Ultralow-temperature preservation and activation method for sperms of sebastes schlegeli hilgendorf of oviparous fishes | |
| CN110999898A (en) | Method for cryopreservation of procypris merus sperm | |
| CN106818708B (en) | Ultralow temperature cryoprotectant for epinephelus coioides semen and preservation method thereof | |
| Riley et al. | Cryopreservation of sperm of red snapper (Lutjanus campechanus) | |
| CN109122668A (en) | A kind of store method of hilsa herring sperm | |
| CN116235847A (en) | Low-temperature cryopreservation tube, and grouper low-temperature cryopreservation method and application | |
| CN112352777A (en) | Sperm cryopreservation liquid and application thereof in sperm cryopreservation of Bostrichthys sinensis | |
| CN101342192B (en) | Quick freezing conservation method for bastard halibut spermatozoon | |
| CN112075415B (en) | Cryoprotectant and ultra-low temperature cryopreservation method for stichopus japonicus sperms | |
| CN113455497B (en) | Ultralow temperature cryopreservation method for sperm of penaeus chinensis | |
| CN100348096C (en) | Vitrifiation freezing preservation method for genine porgy embryo particles | |
| CN104304236A (en) | Erythroculter ilishaeformis semen cryopreservation solution | |
| CN107047540A (en) | A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution | |
| CN112970742A (en) | Efficient ultralow-temperature cryopreservation method for phoxinus tinctoria sperms | |
| CN102986652B (en) | Superfreeze preservation method of pink salmon 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: 20200414 |
|
| RJ01 | Rejection of invention patent application after publication |