CN107047540A - A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution - Google Patents
A kind of Chang Tun Catfish seminal fluid cryopreservation method and its dilution Download PDFInfo
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Abstract
The invention discloses a kind of the preservation dilution and cryopreservation method of Chang Tun Catfish seminal fluid.The preservation dilution is made up of sugar, sodium salt, sylvite, calcium salt, tyrosine, glycine, distilled water and antifreeze dimethyl sulfoxide (DMSO).The cryopreservation method comprises the following steps：Select qualified excellent male parent population to carry out semen collection, be stored in the preservation dilution for having added neomycinsulphate and streptomysin, using 10.5 DEG C/30s rate of temperature fall, after reaching 150 DEG C, be put into liquid nitrogen and carry out cryopreservation；When needing defrosting recovery, Chang Tun Catfish seminal fluid of cryopreservation is taken out, the PBS of volume fraction 0.65%~0.7% is added as thawing solution, quick-thawing in 32 DEG C~40 DEG C of water-bath is put into and recovers.The above-mentioned technology of the present invention ensure that the persistence of Chang Tun Catfish Semen routine times, and good to the protecting effect of seminal fluid, and survival rate height, the vigor of sperm are good after preservation.
Description
Technical field
The invention belongs to technical field of aquaculture.More particularly, to a kind of Chang Tun Catfish of rare easy danger fish seminal fluid
Cryopreservation method and its dilution.
Background technology
Chang Tun Catfish are SILURIFORMESs, Chang Tun Catfish sections, and Chang Tun Catfish belong to fish.Body is long, flat-sided, and dorsal fin starting point is body highest point.Head
Flat flat, slightly triangular in shape, back side bone is coarse exposed.Muzzle goes out, blunt circle.Mouth near-end position, arc, the upper jaw is slightly protruded.For subtropical zone
The foot of a mountain river small stream demersal fishes, like limpid flowing water environment.Chang Tun Catfish between flesh without piercing, and meat flavour is delicious, is of high nutritive value, so on the market
Price is higher, the situation that supply falls short of demand often occurs.Wild Chang Tun Catfish cause its wild resource and seed due to overfishing
It is deficient（Cause the wild resources such as seedling very deficient）.Meanwhile, Chang Tun Catfish are that Pearl River Delta is most rare, artificial breeding difficulty maximum
One of famous-brand and high-quality economic fish.Although Some Domestic scholar is devoted for years in Chang Tun Catfish artificial propagation, fertilization biology
Basic research, making every effort to find influences the key factor of Chang Tun Catfish artificial inseminations seed rate of fertilization survival rate, but has little effect.
Our seminars find the Chang Tun Catfish artificial propagations of restriction on the basis of the Chang preliminary artificial propagations of Tun Catfish of the Zhujiang River are successful
Principal element be not the traditional environment ecological factor such as water temperature, pH, nor female parent population archiblast is bad, problem may go out in hero
It is relevant with the gonad development state of male parent population with parent population, and it is difficult to obtain to develop preferably male parent population, even if obtaining once in a while
Preferably male parent population is developed, but female parent population may also not reach maturity, and it is long stern Catfish that this male and female, which develop nonsynchronous phenomenon,
Artificial propagation can not successful bottleneck problem.Therefore, be to the preservation of Chang Tun Catfish male parent population seminal fluid it is vital, at present still
It there are no research and the report of correlation.
The content of the invention
The technical problem to be solved in the present invention is to overcome the shortcomings of existing Chang Tun Catfish Semen routines technology there is provided a kind of long stern
Catfish semen diluents and cryopreservation method, are the research of its germplasm and genetic management, semen cryopreservation and Conservation etc.
Provide safeguard, to solving Chang Tun Catfish artificial propagation techniques problem, protecting Chang Tun Catfish resources and realizing the famous-brand and high-quality fish farming industry of China
Sustainable health development important scientific guidance is provided.
It is an object of the invention to provide a kind of Chang Tun Catfish Semen routine dilutions.
Another object of the present invention is to provide a kind of long stern Catfish seminal fluid cryopreservation methods.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
A kind of Chang Tun Catfish Semen routine dilutions, by sugar, sodium salt, sylvite, calcium salt, tyrosine, glycine, distilled water and antifreeze
Composition；The antifreeze is dimethyl sulfoxide (DMSO).
Wherein it is preferred to, the sugar is glucose, or the sugar is glucose and fructose.
Preferably, the sodium salt is NaCl, NaHCO3Or Na2HPO4In one or more.
Preferably, the sylvite is KCl, or the sylvite is KCl and KH2PO4。
Preferably, the calcium salt is CaCl2。
Preferably, Chang Tun Catfish Semen routine dilutions also include magnesium salts；Preferably, the magnesium salts is MgCl2Or
MgSO4。
Chang Tun Catfish Semen routines dilution of the present invention adds neomycinsulphate and streptomysin using preceding.Preferably, sulfuric acid
The addition volume ratio of neomycin and streptomysin is 1~1.2：1.
Particularly preferably, Chang Tun Catfish Semen routine dilutions of the present invention have following tri- kinds of formulas of A, B, C：
A is formulated：In per 100mL distilled water containing 0.15~0.2g glucose, 1.5~1.6g NaCl, 0.15~0.18g KCl,
0.02~0.03g CaCl2, 0.04~0.05g MgSO4·7H2O, 0.06~0.07g NaHCO3, 0.03~0.04g
Na2HPO4·H2O, 0.01~0.015g KH2PO4, 0.18~0.2g C6H12O6·H2O, final volume concentration is the two of 5%~15%
Methyl sulfoxide；
B is formulated：Contain 1.5~2g glucose, 1~1.5g fructose, 0.05~0.1g KCl, 0.01 in per 100mL distilled water
~0.03g CaCl2, 0.18~0.25g NaHCO3, final volume concentration is 5%~15% dimethyl sulfoxide (DMSO)；
C is formulated：In per 100mL distilled water containing 2.5~3g glucose, 0.8~1g NaCl, 0.1~0.2g KCl, 0.02~
0.04g CaCl2, 0.01~0.02g MgCl2·6H2O, 0.05~0.08g Na2HPO4, 0.3~0.4g NaHCO3, 0.1~
0.2g tyrosine, 0.05~0.1g glycine, final volume concentration are 5%~15% dimethyl sulfoxide (DMSO).
It is highly preferred that tri- kinds of formulas of A, B, C of Chang Tun Catfish Semen routine dilutions are as follows：
A is formulated：Contain 0.18g glucose, 1.52g NaCl, 0.168g KCl, 0.028g CaCl in per 100mL distilled water2、
0.0472g MgSO4·7H2O、0.065g NaHCO3、0.035g Na2HPO4·H2O、0.012g KH2PO4、0.188g
C6H12O6·H2O, final volume concentration is 10% dimethyl sulfoxide (DMSO)；
B is formulated：Contain 1.8g glucose, 1.2g fructose, 0.08g KCl, 0.02g CaCl in per 100mL distilled water2、
0.21g NaHCO3, final volume concentration is 10% dimethyl sulfoxide (DMSO)；
C is formulated：Contain 2.68g glucose, 0.98g NaCl, 0.16g KCl, 0.03g CaCl in per 100mL distilled water2、
0.012g MgCl2·6H2O、0.06g Na2HPO4、0.32g NaHCO3, 0.12g tyrosine, 0.08g glycine, final volume is dense
Spend the dimethyl sulfoxide (DMSO) for 10%.
Above spermatozoa preservative fluid is standby with salable Brown Glass Brown glass bottles and jars only Cord blood.
In three of the above Semen routine dilution formula of liquid, the most pronounced effects being formulated especially with C.
In addition, a kind of cryopreservation method of Chang Tun Catfish seminal fluid, comprises the following steps：
S1. fingerling is selected：Selection is without disease, the obvious male parent population of sexal maturity feature；
S2. Semen routine dilution is prepared；
S3. collecting semen：Male parent population to step S1 carries out semen collection；
S4. seminal fluid cryopreservation：It will be added in step S2 Semen routine dilution after neomycinsulphate and streptomysin, according to
Seminal fluid：Preserve dilution=1：5~6 volume ratio, the Semen routine that S3 is gathered is in dilution is preserved；Then -10 are used
~-11 DEG C/30s rate of temperature fall, after reaching -150 DEG C, is put into liquid nitrogen and carries out cryopreservation；
S5. thaw and recover：Chang Tun Catfish seminal fluid of cryopreservation is taken out, thawing solution is added, in the water-bath for being put into 32 DEG C~40 DEG C
Quick-thawing is recovered；The thawing solution is the PBS of volume fraction 0.65%~0.7%.
Wherein it is preferred to, fingerling selection described in step S1 is specifically：Profile is first selected without sick and wounded, older（It is ripe
Phase）, it is individual heavier（More than 3 jin）Individual carry out reinforced cultivating；Then the strong health of selection constitution, body surface not damaged, nothing are filled
The illnesss such as blood, abdominal distension, rotten fin, expophthalmos, and sexal maturity feature is obvious, physique healthy and strong, form are rectified, trip appearance is steady, body colour is bright-coloured
Male parent population collecting semen.This method is of crucial importance to the Semen routine in later stage.
Preferably, the specific method of step S3 collecting semens may be referred to as follows：Fish body is dried before semen collection and urine is excluded
And excrement, after being anaesthetized using MS-222 anesthetic to parent population, spermary is taken out in rapid on aseptic operating platform, is weighed, with cutting
Knife, which thoroughly shreds spermary, makes sperm fully discharge, and collects to obtain sperm suspension.Above overall process is operated on ice, sperm suspension
In comprising sperm, spermatid, refining, haemocyte and microtissue fragment etc..
Preferably, neomycinsulphate or streptomysin described in step S4：The volume ratio for preserving dilution is 1~1.2：1.
Preferably, rate of temperature fall described in step S4 is -10.5 DEG C/30s.
Preferably, the container used in step S4 is plastic centrifuge tube, i.e., add step S2 Semen routine dilution
In plastic centrifuge tube, neomycinsulphate and streptomysin are added, according to seminal fluid：Preserve dilution=1：5~6 volume ratio, by S3
The seminal fluid of collection adds plastic centrifuge tube；Then -10.5 DEG C/30s fall off rate is used, will after -150 DEG C of low temperature are reached
Plastic centrifuge tube, which is directly immersed in liquid nitrogen, carries out cryopreservation.
Preferably, thawed described in step S5 recovery temperature be 36 DEG C.
The invention has the advantages that：
Chang Tun Catfish semen diluents and cryopreservation and method for resuscitation that the present invention is provided, ensure that Chang Tun Catfish Semen routines
The persistence of time, seminal fluid cryopreservation technology has greatly facilitated Chang Tun Catfish germ plasm resources exploitation and breeding improvement, in length
There is great theory and realistic meaning in stern Catfish cultivation.First, can by setting up the excellent Chang Tun Catfish of cultivation frozen sperm storehouse
Excellent Chang Tun Catfish former breeding is preserved for a long time, thus avoid fishing for because of transition, environmental pollution and ecological disruption and caused by
Chang Tun Catfish species extinctions or the upper long-term cultivation of production and inbred cause germ plasm resource to be degenerated and variation phenomenon；Secondly, lead to
Sperm super-low temperature preservation is crossed, gonad development can be enable and reproduction period is asynchronous, the cultivation strain of sex reversal and geographic isolation is handed over
Match somebody with somebody, recover germ plasm resource and natural reserve capacity, and can further industrialization and large-scale production, with very considerable ecology
Economic and social benefit.
Specifically, technical scheme has following significant innovation and meaning：
1st, the method for sampling is improved：Using the rare Chang Tun Catfish easily endangered of the Zhujiang River as invention object, the wild of different geographic populations is gathered
Chang Tun Catfish parent populations, the nonsynchronous problem of male and female gonad development is tentatively solved using seminal fluid cryopreservation technology.
2nd, improvement preserves dilution formula of liquid：Protected according to Chang Tun Catfish biology and sexual gland feature and different diluent formula
Deposit the survival rate height of rear sperm, further improvement made to long stern Catfish sperm storages dilution formula of liquid, and have chosen 3 it is excellent
Good formula, effect is good, especially notable with C formulation efficacies.
3rd, semen protective agent is improved：Sperm protective uses a certain proportion of neomycinsulphate and streptomysin complexing agent, takes
For simple penicillin, preferable protective effect is served to sperm.Sperm cryoprotectant concentration is groped, it has been found that addition
10% dimethyl sulfoxide (DMSO) is best to the sperm effect of cryopreservation.
4th, falling temperature method is optimized：Cryopreservation has made larger improvement using fast cooling method step by step on the basis of forefathers,
There is positive meaning to the preservation effect of sperm.
5th, smart thawing solution is frozen in improvement：Chang Tun Catfish freeze the smart thawing solution PBS of volume fraction 0.65%~0.7%, take
It is more preferable to the protecting effect of seminal fluid for physiological saline or distilled water.
Brief description of the drawings
Fig. 1 is the Percoll separating resulting schematic diagrames of seminal fluid.
Fig. 2 is 3 layers of Percoll discontinuous gradient separating resultings of seminal fluid.
Fig. 3 is that Chang Tun Catfish sperm super-low temperature freezings preserve Sperm motility and survival results after 30d.
Fig. 4 is influence of the different recovery temperature to sperm motility.
Fig. 5 is sperm situation after observation recovery under the multiple of microscope 16 × 10.
Embodiment
The present invention is further illustrated below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
Limit in any form.Unless stated otherwise, the reagent of the invention used, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following examples agents useful for same and material are purchased in market.
The Chang Tun Catfish of embodiment 1 Semen routine dilution
1st, spermatozoa preservative fluid dilution A formulas：Liquid A and liquid B is prepared respectively, groping and test according to us, solution A is optimal
It is formulated and is：Glucose 1.80g, NaCl 15.2g, KCl 1.68g, CaCl20.28g, MgSO4·7H2O 0.472g, finally add
Enter 100ml distilled water dissolving；
Second formula of liquid is NaHCO3 0.65g, Na2HPO4·H2O 0.35g, KH2PO4 0.12g, C6H12O6·H2O 1.88g, most
100ml distilled water dissolving is added afterwards.
Take each 10 mL of solution A, second liquid, after mixing plus distilled water is settled to 100 mL, and add final volume concentration for 5%,
10% or 15% dimethyl sulfoxide (DMSO), that is, be configured to sperm storage diluent A.
2nd, sperm storage dilution B is formulated：Glucose 1.80g, fructose 1.2g, KCl 0.08g, CaCl20.02g,
NaHCO30.21g, distilled water 100mL, and add the dimethyl sulfoxide (DMSO) that final volume concentration is 5%, 10% or 15%.
3rd, sperm storage dilution C is formulated：Glucose 2.68g, NaCl 0.98g, KCl 0.16g, CaCl2
0.03g, MgCl2·6H2O 0.012g, Na2HPO40.06g, NaHCO30.32g, tyrosine 0.12g, glycine 0.08g,
Distilled water 100mL, and add the dimethyl sulfoxide (DMSO) that final volume concentration is 5%, 10% or 15%.
By verification experimental verification, the survival rate of sperm is preferable after above-mentioned 3 good formulas are preserved, especially with C formulation efficacies most
Significantly.The volumetric concentration of dimethyl sulfoxide (DMSO) is best with 10% effect.
The Chang Tun Catfish of embodiment 2 seminal fluid cryopreservation method
The invention provides Chang Tun Catfish seminal fluid cryopreservation method, key step is as follows：Fingerling selection, sperm storage dilution
Liquid and antifreeze are prepared, sperm collection and quality observation, seminal fluid cryopreservation, recovery of thawing, sperm motility are observed.Specifically
Operating method is as follows：
1st, fingerling is selected
The fingerling collection of natural environment is the important parent population sources of Chang Tun Catfish, and by long-term field investigation, we introduce collection
Chang Tun Catfish parent populations tail more than 300, wherein profile is selected without sick and wounded, older, more than 3 jin heavier individual tails more than 100 of individual,
Reinforced cultivating is carried out in Qingyuan City Xing Yu Aqua Sciences Inc. scientific research Demonstration Base.Select constitution strong in collecting semen
Health, body surface not damaged, the illness such as no hyperemia, abdominal distension, rotten fin, expophthalmos, and sexal maturity feature substantially, physique healthy and strong, form end
Just, the male parent population that appearance is steady, body colour is bright-coloured is swum.This method is of crucial importance to the Semen routine in later stage.
2nd, sperm storage diluent preparing
According to embodiment 1, sperm storage dilution is prepared.
3rd, sperm collection, separation and quality are observed
（1）Sperm collection selects the salable light-proof centrifuge tubes of 10mL, as sperm storage container after sterilized processing, totally 60
It is individual, every kind of preservation liquid 20, by preservation liquid being added in centrifuge tube carefully, and respectively according to 1~1.2：1 ratio adds sulphur
Sour neomycin and streptomysin, control group are not added with neomycinsulphate and streptomysin, and the number of finishing.
Fish body is dried before semen collection and urine and excrement is excluded, after being anaesthetized using MS-222 anesthetic to parent population, Yu Wu
It is rapid on bacterium operating desk to take out spermary, weigh, thoroughly being shredded spermary with scissors makes sperm fully discharge.It will collect respectively
Sperm suspension is filtered by husky tulle and carries out mark.
Above overall process is operated on ice, and sperm, spermatid, refining, haemocyte and small group are included in sperm suspension
Knit fragment etc..100% isotonic Percoll solution is with Percoll (Pharmacia, Uppsala, Sweden) stostes and 10
× PBS presses 9：1 ratio is mixed and formed.Then empirically demand is diluted subsequent Percoll gradients by 1 × PBS（10%
~90%）, this process using three kinds of specifications sterile centrifugation tube.
（2）Collection carries out microscopy after terminating, and takes 2 μ l seminal fluid to be observed under the microscope, the vigor of sperm is：About
82 % can make quick straight line forward travel, and 9 % make faint curvilinear motion or swing, and 6% original place is spinned, and 3% is motionless, sperm motility rate
For 0.82.
Further using the detection display of computer sperm assistant analysis instrument, the pH value of fresh semen is 7.1.
As shown in Figure 1,3 layers of Percoll discontinuous gradients separating resulting are such as Chang Tun Catfish sperm Percoll separating resultings
Shown in Fig. 2.
4th, seminal fluid low temperature and cryopreservation
（1）Sperm is divided into two parts, a part of sperm is tested for Cord blood, and it is real that second half sperm is used for cryopreservation
Test.Freezing method use plastic centrifuge tube freezing, with refrigerating capacity it is big, easy to operate, it is safe the features such as, available for essence
Liquid freezen protective and sperm bank are set up.
Seminal fluid is taken out, seminal fluid volume is measured, centrifuge tube is placed in equal volume, seminal fluid is 1 with preserving liquid proportional：5~6, this
Method is easy, with low cost, is adapted to the popularization and application in production.
（2）Specifically, we have carried out Cord blood experiment, 4 DEG C of Cord blood results of sperm such as table 1 below institute to sperm
Show：
Table 1 seminal fluid, 4 DEG C of Cord blood results
Note：" ++ " represents that more than 80~90% sperm is quickly moved；"+" represents that 50~60% sperms are quickly moved；"+- " table
Show that 20~30% sperms are quickly moved；"-" represents all dead or a small amount of vibration in situ.
It was found that too fast or excessively slow cooldown rate can cause cellular damage, the survival rate of Chang Tun Catfish sperms is reduced.
Therefore, the seminal fluid of collection is carried out Cord blood by us using fast cooling method step by step, and we are directed to Chang Tun Catfish sperm, specifically
Method is the fall off rate using -10.5 DEG C/30s, after -150 DEG C of low temperature are reached, is directly immersed in liquid nitrogen, this method effect compared with
It is good, sperm, 3 groups of preservation observations of every kind of spermatozoa preservative fluid point can be protected to greatest extent.Our specific practice is by length
Tun Catfish sperm super-low temperatures, which freeze to take out after 30 days, to thaw, and finds 5% dimethyl sulfoxide of addition and 10% dimethyl sulfoxide to freezing after defrosting
The Sperm motility influence difference of preservation is not notable（37.6% and 36.2%）But all it is significantly higher than 15% dimethyl sulfoxide group（24.4%）,
But from the point of view of living spermatozoa percentage, 10% dimethyl sulfoxide keeps 46.6% to be significantly higher than the 37.2% of 5% dimethyl sulfoxide group（Such as Fig. 3）.
In summary, 10% dimethyl sulfoxide (DMSO) of addition is best as antifreeze effect.
5th, thaw and recover
Recovery of thawing is the process of sperm rising temperature for dissolving recovery, is also the process that sperm rejuvenates, the key factor of defrosting is
Thawing solution and thawing rate.
By our experiments repeatedly and grope, Chang Tun Catfish freeze smart thawing solution and delayed with the PBS of volume fraction 0.65%~0.7%
Fliud flushing effect is preferable.
Thawing rate uses quick-thawing method, and concrete operations are the quick-thawings in 32 DEG C~40 DEG C of water-bath.
This method is corresponding with the process of snap frozen, serves good effect.
We compare the influence discovery for analyzing different recovery temperature to Chang Tun Catfish sperm motilities after defrosting, and recovery temperature is
At 36 DEG C, the sperm motility highest after recovery, is 30.66%.Influence such as Fig. 4 institute of the specific difference recovery temperature to sperm motility
Show.
6th, sperm motility is observed
Preservation effect evaluation be activated using sperm after vigor (quick motile accounts for the percentage of total sperm count) as referring to
Mark.First spermatozoa preservative fluid is taken out and shaken up, it is then quick to suction out a droplet with liquid-transfering gun and be placed on slide, microscope 16 ×
Sperm is observed under 10 multiples, it was observed that being activated immediately with half drop distilled water after sperm, its motion conditions is observed and records.We
It was found that, the percentage for making the sperm of straight ahead motion in the visual field is more than 45%, trembles, swings or make faint curve in situ
The sperm 35% or so of motion, completely motionless sperm are less than 20%, these sperm noncapacitation power（The multiple of microscope 16 × 10
Lower observation sperm situation is as shown in Figure 5）.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of Chang Tun Catfish Semen routine dilutions, it is characterised in that by sugar, sodium salt, sylvite, calcium salt, tyrosine, glycine,
Distilled water and antifreeze composition；The antifreeze is dimethyl sulfoxide (DMSO).
2. Chang Tun Catfish Semen routine dilutions according to claim 1, it is characterised in that described sugared for glucose, or institute
It is glucose and fructose to state sugar；The sodium salt is NaCl, NaHCO3Or Na2HPO4In one or more；The sylvite is KCl,
Or the sylvite is KCl and KH2PO4；The calcium salt is CaCl2。
3. Chang Tun Catfish Semen routine dilutions according to claim 1, it is characterised in that also include magnesium salts；The magnesium
Salt is MgCl2Or MgSO4。
4. Chang Tun Catfish Semen routine dilutions according to claim 1, it is characterised in that its formula is：Steamed per 100mL
Contain 0.15~0.2g glucose, 1.5~1.6g NaCl, 0.15~0.18g KCl, 0.02~0.03g CaCl in distilled water2、
0.04~0.05g MgSO4·7H2O, 0.06~0.07g NaHCO3, 0.03~0.04g Na2HPO4·H2O, 0.01~
0.015g KH2PO4, 0.18~0.2g C6H12O6·H2O, final volume concentration is 5%~15% dimethyl sulfoxide (DMSO)；
Or formula is：In per 100mL distilled water containing 1.5~2g glucose, 1~1.5g fructose, 0.05~0.1g KCl,
0.01~0.03g CaCl2, 0.18~0.25g NaHCO3, final volume concentration is 5%~15% dimethyl sulfoxide (DMSO)；
Or formula is：Contain 2.5~3g glucose, 0.8~1g NaCl, 0.1~0.2g KCl, 0.02 in per 100mL distilled water
~0.04g CaCl2, 0.01~0.02g MgCl2·6H2O, 0.05~0.08g Na2HPO4, 0.3~0.4g NaHCO3、0.1
~0.2g tyrosine, 0.05~0.1g glycine, final volume concentration are 5%~15% dimethyl sulfoxide (DMSO).
5. Chang Tun Catfish Semen routine dilutions according to claim 4, it is characterised in that be formulated and be：The distillation per 100mL
Contain 0.18g glucose, 1.52g NaCl, 0.168g KCl, 0.028g CaCl in water2、0.0472g MgSO4·7H2O、
0.065g NaHCO3、0.035g Na2HPO4·H2O、0.012g KH2PO4、0.188g C6H12O6·H2O, final volume concentration is
10% dimethyl sulfoxide (DMSO)；
Or formula is：Contain 1.8g glucose, 1.2g fructose, 0.08g KCl, 0.02g CaCl in per 100mL distilled water2、
0.21g NaHCO3, final volume concentration is 10% dimethyl sulfoxide (DMSO)；
Or formula is：Contain 2.68g glucose, 0.98g NaCl, 0.16g KCl, 0.03g CaCl in per 100mL distilled water2、
0.012g MgCl2·6H2O、0.06g Na2HPO4、0.32g NaHCO3, 0.12g tyrosine, 0.08g glycine, final volume is dense
Spend the dimethyl sulfoxide (DMSO) for 10%.
6. a kind of cryopreservation method of Chang Tun Catfish seminal fluid, it is characterised in that comprise the following steps：
S1. fingerling is selected：Selection is without disease, the obvious male parent population of sexal maturity feature；
S2. Semen routine dilution is prepared：Semen routine dilution is as described in Claims 1 to 5 is any；
S3. collecting semen：Male parent population to step S1 carries out semen collection；
S4. seminal fluid cryopreservation：It will be added in step S2 Semen routine dilution after neomycinsulphate and streptomysin, according to
Seminal fluid：Preserve dilution=1：5~6 volume ratio, the Semen routine that S3 is gathered is in dilution；Then -10~-11 are used
DEG C/30s rate of temperature fall, after reaching -150 DEG C, it is put into liquid nitrogen and carries out cryopreservation；
S5. thaw and recover：Chang Tun Catfish seminal fluid of cryopreservation is taken out, thawing solution is added, in the water-bath for being put into 32 DEG C~40 DEG C
Quick-thawing is recovered；The thawing solution is the PBS of volume fraction 0.65%~0.7%.
7. the cryopreservation method of Chang Tun Catfish seminal fluid according to claim 6, it is characterised in that fingerling described in step S1 is selected
Select specifically：First select profile and carry out reinforced cultivating without the individual that sick and wounded, maturity period, body weight are not less than 3 jin；Then selective body
The strong health of matter, body surface not damaged, without hyperemia, abdominal distension, rotten fin, expophthalmos illness, and sexal maturity feature substantially, physique healthy and strong, shape
State is rectified, the trip male parent population collecting semen that appearance is steady, body colour is bright-coloured.
8. the cryopreservation method of Chang Tun Catfish seminal fluid according to claim 6, it is characterised in that sulfuric acid described in step S4 is new
Mycin or streptomysin：The volume ratio for preserving dilution is 1~1.2：1.
9. the cryopreservation method of Chang Tun Catfish seminal fluid according to claim 6, it is characterised in that cool speed described in step S4
Rate is -10.5 DEG C/30s.
10. the cryopreservation method of Chang Tun Catfish seminal fluid according to claim 6, it is characterised in that characterized in that, step
Thawed described in S5 recovery temperature be 36 DEG C.
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| CN112715534A (en) * | 2021-03-01 | 2021-04-30 | 内江师范学院 | Efficient ultralow-temperature cryopreservation method for spring fish sperms |
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