CN101502256A - Ultra low temperature cryopreservation method for sperm of large yellow crocker and cryopreservation device - Google Patents
Ultra low temperature cryopreservation method for sperm of large yellow crocker and cryopreservation device Download PDFInfo
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- CN101502256A CN101502256A CNA2009100966143A CN200910096614A CN101502256A CN 101502256 A CN101502256 A CN 101502256A CN A2009100966143 A CNA2009100966143 A CN A2009100966143A CN 200910096614 A CN200910096614 A CN 200910096614A CN 101502256 A CN101502256 A CN 101502256A
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Abstract
The present invention discloses a method for freezing sperm of large yellow croaker. The method comprises the steps of preparing antifreezing liquid, collecting spectrum, preparing the mixture liquid of antifreezing liquid and spectrum, separately packaging the mixture liquid, freezing the mixture liquid, etc. The method for freezing sperm of large yellow croaker according to the invention has the advantages of convenient operation, high survival rate of sperm, better freezing storing quality of sperm and relatively higher activity ratio of sperm after freezing. The invention also discloses a freezing device of the method, wherein the freezing device comprises a foam box provided with a cover. Liquid oxygen is provided in the foam box. The inner depth of foam box is 18 centimeter and the wall thickness is 2.5 centimeter. Two long walls of foam box are symmetrically provided with grooves which extend downwards from the box top of foam box. The downwards depths of grooves from the box top of foam box is 0.5 centimeter. The distance from the liquid surface of liquid nitrogen to the groove is 2.5-3.5 centimeter. The device of the invention has the advantages of more convenient operation, prevention of the phenomenon of liquid nitrogen vapor enveloping after cover opening, saving to the freezing time, prevention the phenomenon of dropping into the liquid nitrogen caused by the rolling of pulled straws, etc.
Description
Technical field
The present invention relates to the fish sperm cryopreservation method, be specifically related to frozen method of large yellow crocker sperm ultralow temperature and frozen device.
Background technology
Large yellow Crocker is the famous and precious marine products economic fish of China, the breed kind that southeastern coast is important, and present large yellow Crocker natural resources rareness, the germplasm degradation phenomena appears in cultured large yellow croaker to some extent, influences the healthy sustainable development of its aquaculture.Fish sperm ultralow temperature is frozen to be one of effective way of quality saving; the high-quality sperm super-low temperature freezing of big fish is got up; set up the sperm bank long preservation, significant to the protection of its germ plasm resource, also can provide the elite germplasm material for breed improvement and rearing new variety.
At present, frozen main employing computer-controlled programmed cooling instrument falling temperature method of fish sperm ultralow temperature and simple and easy heat sink substep falling temperature method.Programmed cooling instrument volume is big, operation is complicated, is adapted at using in the laboratory, is inconvenient to be carried to open-air the use; Simple and easy heat sink can be small and light according to instructions for use design voluntarily, cost saving, volume, is convenient for carrying the field and uses.Because the collection of fish sperm ground often in the open air, and is far away from the laboratory, and after the fish sperm collection, its vigor can weaken after 1～2 hour gradually, therefore, the sperm of gathering was adopted simple and easy heat sink substep falling temperature method superfreeze sperm usually.As the Chinese patent publication number is 101088511, open day is on December 19th, 2007, name is called the simple method for ultralow temperature preservation of fish sperm application for a patent for invention and discloses fish sperm through diluted, after the refrigerator inner equilibrium, add antifreeze dimethyl sulfoxide (DMSO) (DMSO), divide then and install in the wheat tubule, the wheat tubule is positioned on the hollow cystosepiment that floats in bubble chamber above the liquid nitrogen, after keeping 20 fens clock times, the wheat tubule is placed liquid nitrogen at least 5 minutes, change the liquid nitrogen container long preservation again over to.It is frozen that this method is suitable for the ultralow temperature of freshwater fish sperm, because the difference on seawater fish sperm and the freshwater fish physiological property, the sperm life of seawater fish is shorter, this method is relatively poor to the freezing preservation applicability of the seawater fish sperm of large yellow Crocker etc., lower as the sperm survival rate, the preservation of sperm is second-rate, and the activity ratio behind the spermatozoa cryopreservation is relatively low.This method is that the wheat tubule that will contain seminal fluid is positioned on the hollow cystosepiment that floats in the bubble chamber above the liquid nitrogen in addition, because cystosepiment is shrouded by the liquid nitrogen steam, see not too clear when up putting the wheat tubule, inconvenient operation, incur loss through delay the frozen time, also take place the wheat tubule to place improper easily and directly fall into operation error in the liquid nitrogen, and the hollow cystosepiment will be put into bubble chamber, require the bubble chamber volume relatively large, we study and have invented simple and easy preferably frozen method of large yellow Crocker and frozen device for this reason.
Summary of the invention
Technical problem to be solved by this invention provides a kind of easy to operate, and the sperm survival rate is higher, and the preservation quality of sperm is better, the frozen method of large yellow crocker sperm ultralow temperature that the activity ratio behind the spermatozoa cryopreservation is higher relatively.
The present invention also provides the frozen device that is used for above-mentioned frozen method, and it is more convenient that this frozen device has operation, and the back liquid nitrogen steam of having avoided uncapping shrouds phenomenon, saves the frozen time, has avoided the wheat tubule to roll and falls into advantage such as liquid nitrogen phenomenon.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: the frozen method of large yellow crocker sperm ultralow temperature comprises the steps:
1) anti frozen liquid preparation: 85～90:10～15 mix dilution and antifreeze by volume, are mixed with anti frozen liquid and place under 4 ℃ of temperature condition preservation stand-by, and antifreeze commonly used is dimethyl sulfoxide (DMSO) (DMSO);
2) sperm collection: select the male large yellow Crocker of gonadal maturation, dry its gonopore body surface on every side, gently press fish belly to obtain seminal fluid, place culture dish or small beaker, be put on the ice bath;
3) preparation of the mixed liquor of anti frozen liquid and seminal fluid: 1:2.5～3.5 by volume, the seminal fluid of above-mentioned steps 2 and the anti frozen liquid of above-mentioned steps 1 are mixed, obtain mixed liquor, balance is 10～15 minutes under 4 ℃ of temperature condition;
4) mixed liquor packing: 0.35～0.45 milliliter of mixed liquor after the balance is loaded in 0.5 milliliter the wheat tubule;
5) mixed liquor is frozen: the wheat tubule of above-mentioned steps 4 is held in the groove of the simple and easy frozen device that fills liquid nitrogen, the distance of the liquid level of described groove and described liquid nitrogen is 2.5～3.5 centimetres, the lid that covers frozen device kept 2.5～3.5 minutes, uncap then described wheat tubule is dropped in the described liquid nitrogen, the lid that covers frozen device again kept 4～6 minutes, uncapped to pull described wheat tubule out and change in the liquid nitrogen container immediately and preserve.
In above-mentioned steps 1, the volume ratio of described dilution and described antifreeze is 86.7:13.3, and the ratio of this dilution and antifreeze is to the effect of the quality of sperm with the frozen large yellow Crocker of better assurance.
Described dilution is for to form by following formulated:
NaCl:7.25 gram KCl:0.38 gram CaCl
2: 0.18 gram
NaHCO
3: 1.00 gram MgSO
47H
2O:0.23 restrains NaH
2PO
42H
2The O:0.41 gram
Glucose: 1.00 grams, with dissolved in distilled water and fixed molten to 1000 milliliters, this dilution has the effect that guarantees its quality preferably to the sperm of frozen large yellow Crocker.
In above-mentioned steps 3, the volume ratio of described seminal fluid and described anti frozen liquid is 1:3, and the ratio of this seminal fluid and anti frozen liquid also has the quality of the frozen back of better assurance sperm.
In above-mentioned steps 5, the distance of the liquid level of described wheat tubule and described liquid nitrogen is 3 centimetres.
The frozen device that the frozen method of above-mentioned large yellow crocker sperm ultralow temperature is used, comprise bubble chamber with cover, described bubble chamber is built-in with liquid nitrogen, be 18 centimetres deeply in the described bubble chamber, wall thickness is 2.5 centimetres, be symmetrically arranged with the downward groove in case top from described bubble chamber on two longwells of described bubble chamber, described groove is 0.5 centimetre from the downward degree of depth in case top of described bubble chamber, and the liquid level of described liquid nitrogen and the distance of described groove are 2.5～3.5 centimetres.
Compared with prior art, the invention has the advantages that the frozen method of large yellow crocker sperm: prepare by anti frozen liquid, sperm is gathered, the preparation of the mixed liquor of anti frozen liquid and seminal fluid, steps such as mixed liquor packing and mixed liquor are frozen, the configuration of this method anti frozen liquid is more reasonable, the ratio of the mixed liquor of anti frozen liquid and seminal fluid is suitable, the incorporation time of seminal fluid and antifreeze is more reasonable, promptly has freeze proof preferably effect, the survival environment that has preferable sperm again, the frozen operation of this method is easier, and the survival rate of cryopreservation sperm is higher, the quality of frozen sperm is better, the activity ratio of frozen back sperm is higher can to reach 90.91 ± 1.33, the relative fertilization rate of sperm that activates also can reach 92.21 ± 1.56, this method cost is also lower simultaneously, and therefore a kind of easy to operate, the sperm survival rate is higher, the preservation quality of sperm is better, the frozen method of large yellow crocker sperm ultralow temperature that the activity ratio behind the spermatozoa cryopreservation is higher relatively.The frozen device that large yellow crocker sperm ultralow temperature is frozen, comprise bubble chamber with cover, bubble chamber is built-in with liquid nitrogen, be 18 centimetres deeply in the bubble chamber, wall thickness is 2.5 centimetres, be symmetrically arranged with the downward groove in case top from bubble chamber on two longwells of bubble chamber, groove is 0.5 centimetre from the downward degree of depth in case top of bubble chamber, and the liquid level of liquid nitrogen and the distance of groove are 2.5～3.5 centimetres; This device be with the wheat tubule put into groove have the operation more convenient, the back liquid nitrogen steam that prevented to uncap shrouds phenomenon, be the quick frozen condition of having created, the lower time-to-live of essence of marine fishes such as object large yellow Crocker has been created suitable frozen device, when placing the wheat tubule, avoid the rolling of wheat tubule simultaneously and fallen into the liquid nitrogen phenomenon, and the groove of this device at interval evenly rationally, when placing the wheat tubule of same quantity, device volume can be much smaller, help saving the usage amount of liquid nitrogen like this, therefore it is more convenient that this frozen device has operation, the back liquid nitrogen steam of having avoided uncapping shrouds phenomenon, save the frozen time, also avoided the wheat tubule to roll and fall into advantage such as liquid nitrogen phenomenon.
Description of drawings
Fig. 1 is the structural representation of the frozen device of the present invention.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
Embodiment 1
The frozen device that large yellow crocker sperm ultralow temperature as shown in Figure 1 is frozen, comprise with cover 2 bubble chamber 1, bubble chamber 1 is built-in with liquid nitrogen 3, length is 16 centimetres in the bubble chamber 1, wide is 12 centimetres, be 18 centimetres deeply, wall thickness is 2.5 centimetres, on two longwells 12 of bubble chamber 1, be symmetrically arranged with and push up 11 downward grooves 4 from the case of bubble chamber 1, it is 0.5 centimetre that groove 4 pushes up the 11 downward degree of depth from the case of bubble chamber 1, wheat tubule 5 just is shelved in the groove 4, and the liquid level 31 of liquid nitrogen 3 is 2.5～3.5 centimetres with the distance of groove 4.
The frozen method of large yellow crocker sperm ultralow temperature, its step is as follows:
1) anti frozen liquid preparation: 86.7:13.3 mixes dilution and antifreeze by volume, is mixed with anti frozen liquid and places under 4 ℃ of temperature condition preservation stand-by, and wherein the component of dilution is as follows:
NaCl:7.25 gram KCl:0.38 gram CaCl
2: 0.18 gram
NaHCO
3: 1.00 gram MgSO
47H
2O:0.23 restrains NaH
2PO
42H
2The O:0.41 gram
Glucose: 1.00 grams, with dissolved in distilled water and fixed molten to 1000 milliliters; Antifreeze is DMSO.
2) sperm collection: the male large yellow Crocker that selects gonadal maturation, dry its gonopore body surface on every side, the light fish belly of pressing obtains seminal fluid, place culture dish or small beaker, be put on the ice bath, stronger as seminal fluid is obtained vigor by the microscopy screening, the activity time, long sperm was used for frozen better like that.
3) preparation of the mixed liquor of anti frozen liquid and seminal fluid: 1:3 by volume, the seminal fluid of above-mentioned steps 2 and the anti frozen liquid of above-mentioned steps 1 are mixed, obtain mixed liquor, balance is 10～15 minutes under 4 ℃ of temperature condition.
4) mixed liquor packing: about 0.4 milliliter after the balance mixed liquor is loaded in 0.5 milliliter the wheat tubule, wheat tubule length is about 13 centimetres, and The faster the better to divide process of assembling.
5) mixed liquor is frozen: the wheat tubule of above-mentioned steps 4 is held in the groove of frozen device of embodiment 1, the distance of the liquid level of groove and liquid nitrogen is 3 centimetres, the lid that covers frozen device kept 3 minutes, uncap then the wheat tubule is dropped in the liquid nitrogen, the lid that covers frozen device again kept 5 minutes, uncapped to pull the wheat tubule out and change in the liquid nitrogen container immediately and preserve.
The activity ratio of the sperm that the present invention is frozen compares mensuration with activating the back with bright smart run duration, life-span and relative fertilization rate, obtain data as shown in table 1 below, the activity ratio that the frozen back of the present invention sperm is described is higher, and quality is better, and fertilization rate is also higher relatively.
Table 1 cryopreservation sperm and bright smart activity ratio, run duration, life-span and the relative fertilization rate comparison sheet after the activation:
Claims (6)
1, the frozen method of large yellow crocker sperm ultralow temperature is characterized in that comprising the steps:
1) anti frozen liquid preparation: 85～90:10～15 mix dilution and antifreeze by volume, are mixed with anti frozen liquid and place under 4 ℃ of temperature condition preservation stand-by;
2) sperm collection: select the male large yellow Crocker of gonadal maturation, dry its gonopore body surface on every side, gently press fish belly to obtain seminal fluid, place culture dish or small beaker, be put on the ice bath;
3) preparation of the mixed liquor of anti frozen liquid and seminal fluid: 1:2.5～3.5 by volume, the seminal fluid of above-mentioned steps 2 and the anti frozen liquid of above-mentioned steps 1 are mixed, obtain mixed liquor, balance is 10～15 minutes under 4 ℃ of temperature condition;
4) mixed liquor packing: 0.35～0.45 milliliter of mixed liquor after the balance is loaded in 0.5 milliliter the wheat tubule;
5) mixed liquor is frozen: the wheat tubule of above-mentioned steps 4 is held in the groove of the simple and easy frozen device that fills liquid nitrogen, the distance of the liquid level of described groove and described liquid nitrogen is 2.5～3.5 centimetres, the lid that covers frozen device kept 2.5～3.5 minutes, uncap then described wheat tubule is dropped in the described liquid nitrogen, the lid that covers frozen device again kept 4～6 minutes, uncapped to pull described wheat tubule out and change in the liquid nitrogen container immediately and preserve.
2, the frozen method of large yellow crocker sperm ultralow temperature as claimed in claim 1 is characterized in that in above-mentioned steps 1, and the volume ratio of described dilution and described antifreeze is 86.7:13.3.
3, the frozen method of large yellow crocker sperm ultralow temperature as claimed in claim 1 or 2 is characterized in that described dilution is for to form by following formulated:
NaCl:7.25 gram KCl:0.38 gram CaCl
2: 0.18 gram
NaHCO
3: 1.00 gram MgSO
47H
2O:0.23 restrains NaH
2PO
42H
2The O:0.41 gram
Glucose: 1.00 grams, with dissolved in distilled water and fixed molten to 1000 milliliters.
4, the frozen method of large yellow crocker sperm ultralow temperature as claimed in claim 1 is characterized in that in above-mentioned steps 3, and the volume ratio of described seminal fluid and described anti frozen liquid is 1:3.
5, the frozen method of large yellow crocker sperm ultralow temperature as claimed in claim 1 is characterized in that in above-mentioned steps 5,
The distance of the liquid level of described wheat tubule and described liquid nitrogen is 3 centimetres.
6, the frozen method of the described large yellow crocker sperm ultralow temperature of claim 1 used frozen device, comprise bubble chamber with cover, described bubble chamber is built-in with liquid nitrogen, it is characterized in that being 18 centimetres deeply in the described bubble chamber, wall thickness is 2.5 centimetres, be symmetrically arranged with the downward groove in case top from described bubble chamber on two longwells of described bubble chamber, described groove is 0.5 centimetre from the downward degree of depth in case top of described bubble chamber, and the liquid level of described liquid nitrogen and the distance of described groove are 2.5～3.5 centimetres.
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Cited By (7)
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| CN101849535A (en) * | 2010-05-24 | 2010-10-06 | 中国水产科学研究院黄海水产研究所 | Portable fish sperm frozen cooling device |
| CN102217590A (en) * | 2011-08-01 | 2011-10-19 | 天津科技大学 | Pomfret sperm cryopreservation method |
| CN102524241A (en) * | 2010-12-16 | 2012-07-04 | 宁波大学 | Additive for freezing storage protective liquid of fish spermatid |
| CN104904708A (en) * | 2015-06-10 | 2015-09-16 | 浙江省海洋水产研究所 | Small yellow croaker sperm cryopreservation fluid and small yellow croaker sperm cryopreservation method |
| CN105230609A (en) * | 2015-11-06 | 2016-01-13 | 刘玉鹏 | Formula of basic protection liquid for fish sperms |
| CN107736339A (en) * | 2017-10-25 | 2018-02-27 | 南京农业大学 | A kind of novel portable temperature regulating device and use its boar semen method |
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| CN1309301C (en) * | 2003-09-28 | 2007-04-11 | 中国水产科学研究院东海水产研究所 | Ultra low temperature refrigeration method for preserving sperm of sturgeon |
| CN1295956C (en) * | 2004-07-27 | 2007-01-24 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
| CN100484397C (en) * | 2006-11-28 | 2009-05-06 | 厦门大学 | Daiquzu pseudosciaena crocea and minyuedongzu cultured pseudosciaena crocea cross breeding method |
| CN101088511B (en) * | 2007-02-15 | 2011-01-12 | 中国水产科学研究院长江水产研究所 | Simple method for ultralow temperature preservation of fish sperm |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101849535A (en) * | 2010-05-24 | 2010-10-06 | 中国水产科学研究院黄海水产研究所 | Portable fish sperm frozen cooling device |
| CN101849535B (en) * | 2010-05-24 | 2013-04-17 | 中国水产科学研究院黄海水产研究所 | Portable fish sperm frozen cooling device |
| CN102524241A (en) * | 2010-12-16 | 2012-07-04 | 宁波大学 | Additive for freezing storage protective liquid of fish spermatid |
| CN102217590A (en) * | 2011-08-01 | 2011-10-19 | 天津科技大学 | Pomfret sperm cryopreservation method |
| CN104904708A (en) * | 2015-06-10 | 2015-09-16 | 浙江省海洋水产研究所 | Small yellow croaker sperm cryopreservation fluid and small yellow croaker sperm cryopreservation method |
| CN104904708B (en) * | 2015-06-10 | 2017-03-29 | 浙江省海洋水产研究所 | Carnis Pseudosciaenae sperm cryopreservation liquid and Carnis Pseudosciaenae sperm cryopreservation method |
| CN105230609A (en) * | 2015-11-06 | 2016-01-13 | 刘玉鹏 | Formula of basic protection liquid for fish sperms |
| CN107736339A (en) * | 2017-10-25 | 2018-02-27 | 南京农业大学 | A kind of novel portable temperature regulating device and use its boar semen method |
| CN109625634A (en) * | 2019-02-14 | 2019-04-16 | 杨柳 | A kind of human embryo's transfer box |
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