CN105794771A - Cryopreservation method of panda semen - Google Patents
Cryopreservation method of panda semen Download PDFInfo
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- CN105794771A CN105794771A CN201610288665.6A CN201610288665A CN105794771A CN 105794771 A CN105794771 A CN 105794771A CN 201610288665 A CN201610288665 A CN 201610288665A CN 105794771 A CN105794771 A CN 105794771A
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- 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
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Abstract
The invention provides a cryopreservation method of panda semen. The cryopreservation method comprises the following steps: collecting panda seminal fluid; putting the seminal fluid into a semen freezing tube and putting the semen freezing tube into a cavity of a program-controlled freezing instrument, wherein the inside temperature of the cavity is 15-24 DEG C; carrying out gradient cooling on the in-cavity temperature of the cavity with the semen freezing tube, wherein the gradient cooling comprises a first cooling phase and a second cooling phase; and putting the semen freezing tube subjected to the gradient cooling into liquid nitrogen and preserving. The method adopts the programmed gradient cooling and is simple to operate; and meanwhile, the semen activity and the freezing recovery rate of the panda seminal fluid can be improved, and the quality of the frozen semen is improved.
Description
Technical field
The present invention relates to animal sperm Techniques of preserving field, in particular to a kind of Micrograph On Spermatoa of The Giant Panda
Freezing and storing method.
Background technology
Giant panda is the distinctive rareness species of China, and fertility is low, and oestrus is short.Big in stable breeding
In panda population, owing to being simultaneously in the male and female giant panda negligible amounts of idiophase, the one-tenth of natural mating
Power is relatively low, so the method for artificial fertilization sometimes can be taked to promote giant panda conceived.Artificial collection
The giant panda seminal fluid arrived, viable sperm therein can gradually lose activity in normal temperature environment, affect people
The conception rate of work fertilization.
Existing giant panda Semen freezing technique is frozen spermatic fluid technology, this technology due to during freezing only
It is only through adjusting the distance between freezer surface and liquid nitrogen surface to determine that the first of seminal fluid freezes temperature.Due to hands
The intermittence of work operation, causes during being dripped on metallic plate by seminal fluid, sperm in same batch
Variant with the time of contact of metallic plate so that same batch freezing of semen effect is inconsistent, and impact is frozen
Deposit the quality of sperm.After recovery, owing to the activity difference of same batch sperm is big, can reduce greatly
The success rate of artificial fertilization.
Summary of the invention
It is an object of the invention to provide the freezing and storing method of a kind of Micrograph On Spermatoa of The Giant Panda, this method energy
Enough improve the quality of cryopreservation sperm, to increase the success rate of panda artificial fertilization further.
In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:
The freezing and storing method of a kind of Micrograph On Spermatoa of The Giant Panda, comprises the following steps:
A. giant panda seminal fluid is collected；
B. being put into by the described seminal fluid of gained in described a step freezes in spermaduct, and the described spermaduct that freezes is put into journey
In the cavity of control frigorimeter, the temperature of described cavity is 15 DEG C-24 DEG C；
C. described cavity is carried out gradient cooling: the first temperature-fall period, with the first cooldown rate by described chamber
The temperature of body is reduced to the first cryogenic temperature, and described first cooldown rate is 89 DEG C/min-109 DEG C/min,
Described first cryogenic temperature is-122 DEG C to-102 DEG C；Second temperature-fall period, with the second cooldown rate by institute
The temperature stating cavity is reduced to the second cryogenic temperature, and described second cooldown rate is with 65 DEG C/min-85 DEG C
/ min, described second cryogenic temperature is-175 DEG C to-155 DEG C；Subsequently the described spermaduct that freezes is put into liquid nitrogen
Preserve.
In prior art, the cryopreservation methods of giant panda seminal fluid mainly has three kinds: granule method, ampoule method and
Frozen pipe method.Wherein, granule method equipment is simple, the most easily makes frozen seminal fluid be contaminated,
Simultaneously because granule exposes overlong time at room temperature during gripping so that answering of cryopreservation sperm
Soviet Union's rate is low.Ampoule method complex operation, seals and seals off and all acquire a certain degree of difficulty, and may be by when sealing
Set off an explosion in uneven in temperature, operator and seminal fluid are caused damage.The operation of frozen pipe method is relatively easy,
But owing to, during manual freezing, the maneuver of different operating person differs, can be to the sperm of giant panda
Motility rate produces impact in various degree.
Compared with prior art, the invention have the benefit that
(1) spermaduct that freezes that will be equipped with giant panda seminal fluid is positioned over the cavity of program-controlled freezing instrument, uses program
Gradient cooling method, reduces cavity inner temperature in two stages with different cooldown rates, simple to operate,
It is easy to Automated condtrol, reduces the impact of manual operation semen quality frozen on panda.
(2) by controlling the cooldown rate of program-controlled freezing instrument cavity inner temperature, make cavity inner temperature with the
One 89 DEG C/min-109 DEG C/min of cooldown rate is reduced to the first cryogenic temperature (-122 DEG C to-102 DEG C)
After, then it is reduced to the second cryogenic temperature (65 DEG C/min with the second 65 DEG C/min-85 DEG C/min of cooldown rate
-85 DEG C/min), this gradient cooling can make the spermatid frozen in spermaduct within the cavity quickly cross over ice
The brilliant phase, it is to avoid produce ice crystal in spermatid, reduce the ice crystal damage to sperm membrane, improve giant panda
The motility of sperm of seminal fluid and freezing recovery rate, improve the quality of cryopreservation sperm.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but this area skill
Art personnel are it will be appreciated that the following example is merely to illustrate the present invention, and are not construed as limiting the present invention
Scope.Unreceipted actual conditions person in embodiment, the condition advised according to normal condition or manufacturer
Carry out.Agents useful for same or instrument unreceipted production firm person, being can be by commercially available purchase acquisition
Conventional products.
The freezing and storing method of a kind of Micrograph On Spermatoa of The Giant Panda that present embodiment provides, comprises the following steps.
Step a. collects giant panda seminal fluid.
In preferred embodiment of the present invention, electrostimulation is e.g. used to collect seminal fluid.Concrete,
After giant panda being anaesthetized with ketamine (7mg/kg), stimulate giant panda with electrode bar.Stimulate electricity
Pressure, from the beginning of 2V, according to the degree of giant panda hind limb withdrawal, is slowly increased stimulation voltage.Stimulate every time
Time is about 2 seconds, and the interval time of stimulation is about 3 seconds.Persistently stimulate until extraction seminal fluid.Gather
To seminal fluid can be diluted with diluent TEST (product of Irvine Scientific), and use glycerol
Cook protective agent.
The seminal fluid of gained in step a is put into and is frozen in spermaduct by step b., and will freeze spermaduct and put into program control cold
Freezing in the cavity of instrument, the cavity inner temperature of cavity is 15 DEG C-24 DEG C.
In preferred embodiment of the present invention, by gained when freezing the cavity that spermaduct puts into program-controlled freezing instrument,
The cavity inner temperature of cavity is preferably 17 DEG C-22 DEG C.When the temperature of cavity is preferably 17 DEG C-22 DEG C,
The impact frozen when spermaduct is transferred to program-controlled freezing instrument Micrograph On Spermatoa of The Giant Panda vigor can be reduced, thus enter one
The quality of the raising cryopreservation sperm of step.
It is further preferred that before being put into by seminal fluid and freezing spermaduct, seminal fluid is in the refrigerating equipment of 2 DEG C-8 DEG C
Place 2-8 hour.Before being put into by seminal fluid and freezing spermaduct, seminal fluid cold preservation in low temperature balances a period of time,
Avoid cell activity decrease at room temperature, help to maintain the activity of spermatid.It is further preferred that
Freeze and sealer after spermaduct installs seminal fluid, can be used to carry out encapsulation process to freezing spermaduct.
Step c. carries out gradient cooling to cavity: the first temperature-fall period, with the first cooldown rate by cavity
Temperature be reduced to the first cryogenic temperature, the first cooldown rate is 89 DEG C/min-109 DEG C/min, first
Cryogenic temperature is-122 DEG C to-102 DEG C；Second temperature-fall period, with the second cooldown rate by the temperature of cavity
Being reduced to the second cryogenic temperature, the second cooldown rate is with 65 DEG C/min-85 DEG C/min, and second is freezing
Temperature is-175 DEG C to-155 DEG C；Spermaduct will be frozen subsequently put into liquid nitrogen and preserve.
In preferred embodiment of the present invention, the first cooldown rate in the first temperature-fall period is preferably
94℃/min-104℃/min.When the first cooldown rate is 94 DEG C/min-104 DEG C/min, freeze spermaduct
Interior spermatid with the speeds cross ice crystal phase faster, thus can protect spermatid from ice crystal
Destruction.
It is further preferred that the first cooldown rate in the first temperature-fall period is 92 DEG C/min-98 DEG C/min.
When the first cooldown rate is 92 DEG C/min-98 DEG C/min, freezing the spermatid in spermaduct can be with comparatively fast
The speeds cross ice crystal phase, reduce the quantity of intracellular formation ice crystal, thus reduce ice to greatest extent
The brilliant damage to spermatid film, is conducive to improving the quality of cryopreservation sperm.
In preferred embodiment of the present invention, the first cryogenic temperature in the first temperature-fall period is preferably
-117 DEG C to-107 DEG C.After first temperature-fall period terminates, in program-controlled freezing instrument, the cavity inner temperature of cavity is
-117 DEG C to-107 DEG C, advantageously reduce the damage of spermatid, improve the cell after cryopreservation sperm is thawed
Vigor.
The second cooling speed further, in preferred embodiment of the present invention, in the second cooling stage
Rate is 70 DEG C/min-80 DEG C/min.Second cooldown rate is little compared with the first cooldown rate, and cavity inner temperature exists
During the second cooling stage, cooling is relatively mild, when the second cooldown rate is 70 DEG C/min-80 DEG C/min
Time, seminal fluid variations in temperature in the environment relatively small to the damage of spermatid.Second cooling speed
Rate more preferably 73 DEG C/min-78 DEG C/min, now the variations in temperature in cavity is to spermatid
Degree of injury is minimum.
In preferred embodiment of the present invention, the second cryogenic temperature in the second temperature-fall period is preferably
-170 DEG C to-160 DEG C.After second temperature-fall period terminates, in program-controlled freezing instrument, the cavity inner temperature of cavity is
-170 DEG C to-160 DEG C, advantageously reduce the damage of spermatid, improve the cell after cryopreservation sperm is thawed
Vigor, improves the quality of cryopreservation sperm further.
Embodiment 1
The freezing and storing method of a kind of Micrograph On Spermatoa of The Giant Panda, comprises the following steps:
1. giant panda semen collection: after giant panda being anaesthetized with ketamine (7mg/kg), electricity consumption
Extremely rod stimulates giant panda.Stimulation voltage is from the beginning of 2V, according to the degree of giant panda hind limb withdrawal, slowly
Increase stimulation voltage.Stimulation time 2 seconds every time, the interval time of stimulation is 3 seconds.Persistently stimulate straight
To extraction seminal fluid.
2. the seminal fluid diluent TEST (product of Irvine Scientific) collected is diluted.
In diluent TEST, add cryoprotective agent glycerol before dilution, thus reduce and cause owing to ice crystal is formed
Cell injury.In seminal fluid after dilution, the density of sperm is 5 × 108/ml, i.e. dilution after every
Milliliter seminal fluid there are 5 × 108 spermatids..Above-mentioned seminal fluid is positioned in 4 DEG C of refrigerators balance 4 little
Shi Hou, sucks seminal fluid and freezes in spermaduct, uses sealer to carry out encapsulation process to freezing spermaduct after installing.
Then in the cavity freezing spermaduct and putting into program-controlled freezing instrument, now in the cavity of the cavity of program-controlled freezing instrument
Temperature is 19 DEG C.
3. start the freezing procedure of program-controlled freezing instrument.Program-controlled freezing instrument is first with the cooling speed of 89 DEG C/min
Rate is reduced to-122 DEG C the cavity inner temperature of cavity, subsequently with the cooldown rate of 65 DEG C/min cavity
Cavity inner temperature be reduced to-165 DEG C after, terminate freezing, will freeze spermaduct and take out and put in liquid nitrogen preservation.
Embodiment 2
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 1 are basically identical, and difference is, in program-controlled freezing instrument, the parameter of freezing procedure sets
Put.
Starting the freezing procedure of program-controlled freezing instrument, program-controlled freezing instrument is first with the cooling speed of 94 DEG C/min
Rate is reduced to-117 DEG C the cavity inner temperature of cavity, subsequently with the cooldown rate of 70 DEG C/min cavity
Cavity inner temperature be reduced to-170 DEG C after, terminate freezing, will freeze spermaduct and take out and put in liquid nitrogen preservation.
Embodiment 3
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 1 are basically identical, and difference is, in program-controlled freezing instrument, the parameter of freezing procedure sets
Put.
Starting the freezing procedure of program-controlled freezing instrument, program-controlled freezing instrument is first with the cooling speed of 99 DEG C/min
Rate is reduced to-112 DEG C the cavity inner temperature of cavity, subsequently with the cooldown rate of 75 DEG C/min cavity
Cavity inner temperature be reduced to-165 DEG C after, terminate freezing, will freeze spermaduct and take out and put in liquid nitrogen preservation.
Embodiment 4
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 1 are basically identical, and difference is, in program-controlled freezing instrument, the parameter of freezing procedure sets
Put.
Starting the freezing procedure of program-controlled freezing instrument, program-controlled freezing instrument is first with the cooling speed of 104 DEG C/min
Rate is reduced to-107 DEG C the cavity inner temperature of cavity, subsequently with the cooldown rate of 80 DEG C/min cavity
Cavity inner temperature be reduced to-160 DEG C after, terminate freezing, will freeze spermaduct and take out and put in liquid nitrogen preservation.
Embodiment 5
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 1 are basically identical, and difference is, in program-controlled freezing instrument, the parameter of freezing procedure sets
Put.
Starting the freezing procedure of program-controlled freezing instrument, program-controlled freezing instrument is first with the cooling speed of 109 DEG C/min
Rate is reduced to-102 DEG C the cavity inner temperature of cavity, subsequently with the cooldown rate of 85 DEG C/min cavity
Cavity inner temperature be reduced to-155 DEG C after, terminate freezing, will freeze spermaduct and take out and put in liquid nitrogen preservation.
Embodiment 6
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 3 are basically identical, and difference is: will freeze spermaduct when putting into program-controlled freezing instrument, journey
The cavity inner temperature of the cavity of control frigorimeter is 15 DEG C.
Embodiment 7
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 3 are basically identical, and difference is: will freeze spermaduct when putting into program-controlled freezing instrument, journey
The cavity inner temperature of the cavity of control frigorimeter is 24 DEG C.
Embodiment 8
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 3 are basically identical, and difference is: will collect seminal fluid by electrostimulation, dilution
After be positioned in 2 DEG C of ice baths cooling 2 hours after, load freeze spermaduct.
Embodiment 9
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda that the present embodiment provides, implementation step and specifically joining
Number and embodiment 3 are basically identical, and difference is: will collect seminal fluid by electrostimulation, dilution
After be positioned in 8 DEG C of ice baths cooling 8 hours after, load freeze spermaduct.
Reference examples 1
Use traditional straw frozen semen technology that giant panda seminal fluid is preserved.1. giant panda semen collection:
After giant panda being anaesthetized with ketamine (7mg/kg), stimulate giant panda with electrode bar.Stimulate electricity
Pressure, from the beginning of 2V, according to the degree of giant panda hind limb withdrawal, is slowly increased stimulation voltage.Stimulate every time
2 seconds time, the interval time of stimulation is 3 seconds.Persistently stimulate until extraction seminal fluid.
2. the seminal fluid diluent TEST (product of Irvine Scientific) collected is diluted.
Adding glycerol before dilution in diluent TEST, glycerol can reduce intracellular ice crystal as protective agent
Formed, thus reduce owing to ice crystal forms the cell injury caused.In seminal fluid after dilution, sperm is close
Degree is 5 × 108/ml, i.e. has 5 × 108 spermatids in every milliliter of seminal fluid after dilution.By above-mentioned
Seminal fluid is positioned in 4 DEG C of refrigerators after balancing 4 hours, seminal fluid is sucked and freezes in spermaduct, use close after installing
Envelope machine carries out encapsulation process to freezing spermaduct.
3. first test tube rack is fixed on the position near liquid nitrogen container, then the spermaduct upper strata at test tube rack will be frozen
Placing 1 minute, the distance now freezing spermaduct distance liquid nitrogen surface is 7.5cm；Spermaduct will be frozen the most again turn
Moving to the lower floor of test tube rack, place 1 minute, the distance now freezing spermaduct distance liquid nitrogen surface is 2.5cm.
Reference examples 2
1. giant panda semen collection: after giant panda being anaesthetized with ketamine (7mg/kg), electricity consumption
Extremely rod stimulates giant panda.Stimulation voltage is from the beginning of 2V, according to the degree of giant panda hind limb withdrawal, slowly
Increase stimulation voltage.Stimulation time 2 seconds every time, the interval time of stimulation is 3 seconds.Persistently stimulate straight
To extraction seminal fluid.
2. the seminal fluid diluent TEST (product of Irvine Scientific) collected is diluted.
In diluent TEST, add cryoprotective agent glycerol before dilution, thus reduce and cause owing to ice crystal is formed
Cell injury.In seminal fluid after dilution, the density of sperm is 5 × 108/ml, i.e. dilution after every
Milliliter seminal fluid there are 5 × 108 spermatids.Above-mentioned seminal fluid is positioned in 4 DEG C of refrigerators balance 4 hours
After, seminal fluid is sucked and freezes in spermaduct, after installing, use sealer to carry out encapsulation process to freezing spermaduct.So
Afterwards in the cavity freezing spermaduct and putting into program-controlled freezing instrument, now the cavity temperature of program-controlled freezing instrument is 13 DEG C.
3. start the freezing procedure of program-controlled freezing instrument.Program-controlled freezing instrument is first with the cooling speed of 88 DEG C/min
Rate is reduced to-123 DEG C the temperature in cavity, subsequently with the cooldown rate of 64 DEG C/min in cavity
After temperature is reduced to-166 DEG C, terminate freezing, spermaduct taking-up will be frozen and put into preservation in liquid nitrogen.
Reference examples 3
1. giant panda semen collection: after giant panda being anaesthetized with ketamine (7mg/kg), electricity consumption
Extremely rod stimulates giant panda.Stimulation voltage is from the beginning of 2V, according to the degree of giant panda hind limb withdrawal, slowly
Increase stimulation voltage.Stimulation time 2 seconds every time, the interval time of stimulation is 3 seconds.Persistently stimulate straight
To extraction seminal fluid.
2. the seminal fluid diluent TEST (product of Irvine Scientific) collected is diluted.
In diluent TEST, add cryoprotective agent glycerol before dilution, thus reduce and cause owing to ice crystal is formed
Cell injury.In seminal fluid after dilution, the density of sperm is 5 × 108/ml, i.e. dilution after every
Milliliter seminal fluid there are 5 × 108 spermatids.Above-mentioned seminal fluid is positioned in 4 DEG C of refrigerators balance 4 hours
After, seminal fluid is sucked and freezes in spermaduct, after installing, use sealer to carry out encapsulation process to freezing spermaduct.So
Afterwards in the cavity freezing spermaduct and putting into program-controlled freezing instrument, now the cavity temperature of program-controlled freezing instrument is 25 DEG C.
3. start the freezing procedure of program-controlled freezing instrument.Program-controlled freezing instrument is first with the cooling speed of 110 DEG C/min
Rate is reduced to-101 DEG C the temperature in cavity, subsequently with the cooldown rate of 86 DEG C/min in cavity
After temperature is reduced to-154 DEG C, terminate freezing, spermaduct taking-up will be frozen and put into preservation in liquid nitrogen.
Experimental example
It is respectively adopted through embodiment 1-9 (experimental group) and reference examples 1-3 (matched group) freezen protective
Giant panda seminal fluid carries out controlled trial.It should be noted that embodiment 1-9 (experimental group) and reference examples
In 1-3 (matched group), the age of giant panda, body weight as experimental subject are basically identical.
By freezing of processing through embodiment 1-9 and reference examples 1-3 after spermaduct puts into 37 DEG C of water-bath 30s,
Cut off and freeze one end that spermaduct seals, the seminal fluid frozen in spermaduct is positioned over the diluent Hams of 37 DEG C
In (product of Irvine Scientific), carry out semen analysis with microscope.As a comparison, carrying out
Before above-mentioned freezing processing, the seminal fluid just gathered is put in 37 DEG C of water-baths, carry out semen analysis.
Sperm quality motility of sperm and freezing recovery rate are evaluated, wherein,
Motility of sperm (%): on sperm chromosome drop microscope slide under the conditions of constant temperature is stored in 37 DEG C,
A random selected area count 100, the sperm of every activity is designated as great-hearted sperm, 100
In sperm, the quantity of great-hearted sperm is motility of sperm, and unit is %.
Motility of sperm × 100% before motility of sperm/freezing after Cryopreservation rate=freezing
The sperm quality of table 1 experimental group and matched group compares
As can be seen from Table 1:
1. compared to reference examples 1 uses traditional straw frozen semen technology to preserve giant panda seminal fluid, this
The method (embodiment 1-9) of bright offer effectively raises the motility of sperm of giant panda, and (experimental group is
71%-81%, matched group is 56%-60%), improve the sperm freezing anabiosis rate (experimental group of giant panda
For 82.6%-94.2%, matched group is 63.6%-68.9%), and then conception rate during raising artificial fertilization.
Additionally, this method can effectively reduce the operation impact on motility of sperm, make between different batches
The activity difference of sperm is greatly reduced, and decreases the people caused because of spermatozoon activity difference between different batches
Work is inseminated unsuccessfully probability.
2. compared to the freezing and storing method of the Micrograph On Spermatoa of The Giant Panda used in reference examples 2 and reference examples 3, this
The parameter limited in invention has vital impact to sperm anabiosis rate.Such as, reference examples 2 He
In reference examples 3, the parameters in the freezing procedure of program-controlled freezing instrument is arranged on the ginseng that the present invention limits
Outside number scope, then sperm anabiosis rate is strongly reduced to 63.6%-68.9% by 82.6%-94.2%.
3. in comparative experiments group, in embodiment 1-5, motility of sperm and Cryopreservation rate after freezing can be sent out
Existing, in embodiment 3, motility of sperm (81% ± 3.5) and Cryopreservation rate (94.2%) after freezing are equal
Apparently higher than the motility of sperm (74%-78%) after the freezing of other experimental grouies and Cryopreservation rate
(85.0%-88.6%).Parameters when in step c is described: the first cooldown rate be 99 DEG C/min,
First cryogenic temperature is-112 DEG C, the second cooldown rate is 75 DEG C/min, the second cryogenic temperature is-165 DEG C
Time, motility of sperm and Cryopreservation rate after giant panda freezing are improved significantly.
4. embodiment 1,2,4 and 5 in comparative test group, it is found that great Xiong in embodiment 2 and 4
Motility of sperm (77% and 78%) and Cryopreservation rate (87.5%-88.6%) after cat freezing are above
Motility of sperm (74%-75%) after the freezing of embodiment 1 and embodiment 5 and Cryopreservation rate
(85.0%-86.0%).Illustrate when the parameters in step c: the first cooldown rate is 94 DEG C
/ min-104 DEG C/min, the first cryogenic temperature for-117 DEG C to-107 DEG C, the second cooldown rate be 70 DEG C
/ min-80 DEG C/min, the second cryogenic temperature is when being-160 DEG C to-170 DEG C, after improve giant panda freezing
Motility of sperm and Cryopreservation rate.
5. embodiment 6,7 and embodiment 3 in comparative experiments group, it is found that in b step, journey
When the temperature of control frigorimeter cavity is 19 DEG C, the motility of sperm (81% ± 3.5) after freezing and Cryopreservation
Rate (94.2%) is above the analog value in embodiment 6 and 7, and the sperm in embodiment 6 and 7 is lived
Power (respectively 77% and 78%) and Cryopreservation rate (respectively 89.5% and 88.6%) are the most obvious
Analog value higher than matched group.Illustrate when the temperature of program-controlled freezing instrument cavity is 15 DEG C-24 DEG C, it is possible to
Motility of sperm after raising giant panda freezing and Cryopreservation rate, preferably 19 DEG C.
6. embodiment 8,9 and embodiment 3 in comparative experiments group, it is found that in b step,
Being put into by seminal fluid before freezing spermaduct, seminal fluid is placed 4 hours in the low temp freezing appts of 4 DEG C, and giant panda is cold
Motility of sperm (81% ± 3.5) and Cryopreservation rate (94.2%) after freezing are above embodiment 8 and 9
In analog value, and the motility of sperm (respectively 76% and 71%) in embodiment 8 and 9 and freezing
Anabiosis rate (respectively 82.6% and 87.4%) is still apparently higher than the analog value of matched group.Illustrate inciting somebody to action
Seminal fluid is put into before freezing spermaduct, places 2-8 hour in the low temp freezing appts of 2 DEG C-8 DEG C, it is possible to effectively
Improve the motility of sperm after giant panda freezing and Cryopreservation rate.Wherein, preferably low at 4 DEG C of seminal fluid
Temperature refrigerating equipment is placed 4 hours.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that and do not carrying on the back
May be made that in the case of the spirit and scope of the present invention many other change and amendment.Therefore,
This means all these changes including belonging in the scope of the invention in the following claims and repair
Change.
Claims (10)
1. the freezing and storing method of a Micrograph On Spermatoa of The Giant Panda, it is characterised in that comprise the following steps:
Collect giant panda seminal fluid；
Described seminal fluid is put into and freezes in spermaduct, and the described spermaduct that freezes is put into the cavity of program-controlled freezing instrument,
The cavity inner temperature of described cavity is 15 DEG C-24 DEG C；
The described cavity inner temperature of the described cavity freezing spermaduct described in being placed with is carried out gradient cooling, institute
State gradient cooling and include that the first temperature-fall period and the second temperature-fall period, described first temperature-fall period are with
Described cavity inner temperature is reduced to the first cryogenic temperature by one cooldown rate, and described first cooldown rate is
89 DEG C/min-109 DEG C/min, described first cryogenic temperature is-122 DEG C to-102 DEG C, described second cooling
Stage is with the second cooldown rate described cavity inner temperature to be reduced to the second cryogenic temperature, described second
Cooldown rate is 65 DEG C/min-85 DEG C/min, and described second cryogenic temperature is-175 DEG C to-155 DEG C；
And liquid nitrogen will be put into preserve through freezing spermaduct described in described gradient cooling.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
When will be equipped with freezing described in described seminal fluid the described cavity that spermaduct puts into described program-controlled freezing instrument, described chamber
Body temperature is 17 DEG C-22 DEG C.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
Described first cooldown rate is 94 DEG C/min-104 DEG C/min.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 3, it is characterised in that
Described first cooldown rate is 92 DEG C/min-98 DEG C/min.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
Described first cryogenic temperature is-117 DEG C to-107 DEG C.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
Described second cooldown rate is 70 DEG C/min-80 DEG C/min.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
Described second cooldown rate is 73 DEG C/min-78 DEG C/min.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
Described second cryogenic temperature is-170 DEG C to-160 DEG C.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
Before freezing spermaduct described in being put into by described seminal fluid, described seminal fluid is placed 2-8 hour at 2 DEG C-8 DEG C.
The freezing and storing method of Micrograph On Spermatoa of The Giant Panda the most according to claim 1, it is characterised in that
The method collecting described seminal fluid is electrostimulation.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107173381A (en) * | 2017-06-07 | 2017-09-19 | 成都大熊猫繁育研究基地 | A kind of dry tank freezing method of giant panda seminal fluid |
| CN107211994A (en) * | 2017-06-07 | 2017-09-29 | 成都大熊猫繁育研究基地 | A kind of semen collection of giant panda field and the method for freezing of semen |
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| CN102246744A (en) * | 2011-05-03 | 2011-11-23 | 陕西省动物研究所 | Giant panda semen freezing and preserving fluid and preparation method thereof |
| CN103120156A (en) * | 2013-03-06 | 2013-05-29 | 成都大熊猫繁育研究基地 | New method for preparing frozen semen of giant panda |
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| CN102246744A (en) * | 2011-05-03 | 2011-11-23 | 陕西省动物研究所 | Giant panda semen freezing and preserving fluid and preparation method thereof |
| CN103120156A (en) * | 2013-03-06 | 2013-05-29 | 成都大熊猫繁育研究基地 | New method for preparing frozen semen of giant panda |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107173381A (en) * | 2017-06-07 | 2017-09-19 | 成都大熊猫繁育研究基地 | A kind of dry tank freezing method of giant panda seminal fluid |
| CN107211994A (en) * | 2017-06-07 | 2017-09-29 | 成都大熊猫繁育研究基地 | A kind of semen collection of giant panda field and the method for freezing of semen |
| CN107173381B (en) * | 2017-06-07 | 2020-10-27 | 成都大熊猫繁育研究基地 | Giant panda semen dry tank freezing method |
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| CN105794771B (en) | 2018-09-28 |
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