CN101843238A - Ultralow temperature cryopreservation method of macaca primate semen - Google Patents
Ultralow temperature cryopreservation method of macaca primate semen Download PDFInfo
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- CN101843238A CN101843238A CN201010198716A CN201010198716A CN101843238A CN 101843238 A CN101843238 A CN 101843238A CN 201010198716 A CN201010198716 A CN 201010198716A CN 201010198716 A CN201010198716 A CN 201010198716A CN 101843238 A CN101843238 A CN 101843238A
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Abstract
The invention relates to an ultralow temperature cryopreservation method of macaca primate semen, belonging to an animal breeding method and comprising the following steps: dissolving Tris, TES, lactose, glucose, raffinose, sodiumpenicillinG, streptomycin sulphate and the like in water, centrifuging, taking supernate, preparing the supernate into semen diluent, adding glycerol to prepare deicing fluid, diluting the semen by using diluent, cooling the temperature to 4 DEG C, mixing with the dicing fluid precooled to 4 DEG C with the same volume, subpackaging into frozen straws, cooling by utilizing liquid nitrogen steam, and placing in liquid nitrogen at the temperature of minus 196 DEG C for preservation. When in use, the semen is defrosted and revived. The method in the invention can improve the revived motion range, the survival time, the plasma membrane and acrosome completeness and the fertility rate of the frozen sperm of the macaca primate.
Description
Technical field
The invention belongs to the method for animal reproduction, particularly primate sperm store method.
Background technology
Since Britain biologist Polge etc. with glycerine as antifreezing agent since the freezing preservation sperm; the application frozen sperm carries out in vitro fertilization and artificial insemination is widely used in the treatment of human barrenness and the breed of livestock animals, also provides an approach for the protection of rare species genetic diversity.Macaca primate, as macaque, machin, Assamese macaque, macaca thibetana etc. is human close relative, in institutional framework, aspects such as Physiology and biochemistry, immunity and metabolism are highly similar to the mankind, are the best laboratory animal of setting up researchs such as communicable disease model, spiritual neurogenic disease model, angiocardiopathy model, tumor model and reproductive physiology such as virus.Along with first birth of carrying the transgenosis macaque and the Huntington transgenosis macaque of green fluorescent protein in the world, using primate model research and treating human great functional disease becomes very a hot topic and an important field of research.Each primate species of Macaca is listed in national I and II protection rare species; but on the one hand; the mankind are subjected to various great functional difficult diseases and constantly perplex; pressing for a large amount of macaque animals is used for physicism and solves human health problems; on the other hand; along with continuing to destroy the environmental deterioration that is caused, wild primate field survivorship condition forfeiture descends its fertility.Biological theoretical according to modern reproduction, the sperm of a buck can obtain thousands of offspring's individualities, therefore, efficiently preserves the great number burden that animal sperm can significantly reduce maintenance living animal individuality and population, shortens the breeding cycle, enhances productivity.
With respect to the mankind and livestock animals, the variation to temperature and freezing conditions in refrigerating process of macaca primate sperm is highly brittle weak and responsive, the percentage of head rice of the locomitivity of frozen sperm, plasmalemmae of sperms and acrosome sharply descends, and the sperm time-to-live shortens, fertilization rate in vitro fertilization and test-tube pregnancy rate and farrowing rate are low.The key of percentage of head rice, time-to-live and the fertility of revived motion, plasma membrane and the acrosome of the freezing preservation sperm of raising primate is the development of anti-icing fluid and the design of frozen cooling program.
Glycerine is the most frequently used permeability antifreezing agent of the freezing preservation of animal sperm, and glycerine can infiltrate cell, replaces intracellular hydrone, causes cell dehydration, reduces the formation of intracellular ice crystal in the refrigerating process; Glycerine can form static with the hydrophilic radical of plasma membrane phosphatide and combine, and plays the effect of membrane stabilizer.The concentration of glycerine is the important parameter that influences sperm freezing; the too low purpose that does not reach cryoprotection of concentration; excessive concentration can cause the sperm index to descend; and the sperm of different plant species is to the tolerance difference of glycerine, and the concentration of glycerine is up to 10-20% during as the kangaroo sperm freezing; pig sperm glycerol concentration surpasses 3% damage that can be subjected to because of acrosome and loses fertility; the ox sperm uses the glycerine of 4-8% usually, thereby, the essential the suitableeest glycerol concentration of these species of selecting of different plant species.
Glucide is to be used for the impermeability antifreezing agent that sperm freezing is preserved, except regulating osmotic pressure, make cell dehydration, the hydrophilic phosphate group of the oh group that reduces the formation of intracellular ice crystal and sugar and plasmalemmae of sperms phosphatide combines, stablize outside the plasmalemmae of sperms, freezing point and replacement film on every side the hydrone of carbohydrate by reducing solution suppresses born of the same parents and freezes outward, shorten the dangerous temperature district, reduce the damage that the ice-crystal growth cell membrane causes.In addition, carbohydrate also can influence in the refrigerating process between the shape, ice crystal of the mode of freezing, ice crystal the mechanical attributes of frozen solution width of channel not and frozen solution, therefore, can reduce freezing damage to sperm.And glycan molecule produces three-dimensional effect around being combined in plasma membrane in freezing recovery process, and the film due to the plasma membrane that can stop cell shrinkage to cause is out of shape, folds merges.Equally, carbohydrate to different plant species the cryoprotection effect there are differences, particularly the anti-icing fluid of the glycerine of cryoprotection macaque sperm and the most suitable cooperation concentration of carbohydrate does not also have report.
Summary of the invention
The present invention seeks to deficiency at art methods, a kind of seminal fluid cryopreservation method that is widely used in each primate species of Macaca is provided, the anti-icing fluid that bag expansion glycerine and carbohydrate constitute, and use anti-icing fluid and carry out the method that the seminal fluid superfreeze is preserved and recovered.
The object of the invention realizes by following steps:
(1) get 0.2g Tris, 1.2g TES, 2g lactose, 2g glucose, 0.2g raffinose, 10000IU penicillin G sodium salt, the 5mg streptomycin sulphate is dissolved in the 60mL Milli-Q ultra-pure water successively, add the 20mL egg yolk, mix Hou Jiashui and be settled to 100mL;
(2) with solution centrifugal 1 hour, collect supernatant and make dilution with 7000g;
(3) glycerine of getting 45mL dilution adding 5mL is made anti-icing fluid, is chilled to 4 ℃ in advance;
(4) at room temperature, seminal fluid is mixed in test tube, seals by 1: 9 volume ratio with dilution,, put into the slow cooling of 4 ℃ of refrigerators balance again 2 hours with room-temperature water bath;
(5) in test tube, add isopyknic anti-icing fluid, divide in the straw of packing into after mixing and in liquid nitrogen, preserve; Divide in the 0.25mL straw of packing into after mixing and in liquid nitrogen, preserve;
(6) freezing straw was thawed 2 minutes by being transferred to rapidly in the liquid nitrogen in 37 ℃ the water-bath during recovery.
The room temperature of described method step (4) is 22-25 ℃.
Described method step (5) is to divide anti-icing fluid five times to add test tube, add 1/5 volume at every turn, and each 5 minutes at interval, the final concentration of glycerine was 5%.
Described method step (5) is that straw was placed 5cm place, liquid nitrogen surface top freezing 10 minutes, and rate of temperature fall is 30 ℃/min, preserves in liquid nitrogen again.
Described method step (5) straw volume is 0.25mL.
Tris and TES are respectively the english abbreviation of trishydroxymethylaminomethane and N-three (methylol) methyl-2-aminoethane sulphonic acid sodium.
The good effect that the present invention has is:
The glycerol concentration that the present invention chooses has remarkable result for percentage of head rice, the time-to-live of the revived motion, plasma membrane and the acrosome that improve the macaca primate frozen sperm.
Glucose in the anti-icing fluid of the present invention can to a certain degree pass through cell membrane and enter sperm, and provides metabolisable energy for the low sperm of ATP synthesis capability.C in lactose and the raffinose molecular structure: the H ratio is low, and the quantity of oh group is more than monose, and cell membrane that can the better protection sperm significantly improves the cryoprotection effect to the macaque sperm.
In the semen freezing temperature-fall period, behind the dilution seminal fluid prior to 4 ℃ of cooling balances 2 hours, divide and add the anti-icing fluid that contains glycerine for 5 times and finally reach 5% glycerine final concentration, this pattern can alleviate effectively because the damage that the acute variation that the adding of glycerine causes cell volume to cause because of environment osmotic pressure raises is subjected to, and in the anti-icing fluid of precooling glycerine toxic and side effect 4 ℃ also a lot of with respect to having weakened under the room temperature, this method helps the survival of sperm and the maintenance of cell structure and function.In addition, cooling pattern, rate of temperature fall and the cooling time in the liquid nitrogen steam have also constituted the notable feature of anti-icing fluid result of use of the present invention.
The inventive method is simple, draw materials easily and cost price cheap, do not need special installation, be easy to promote.
Experimental result shows; method of the present invention can improve the revived motion of the various primate frozen sperms of Macaca effectively; protect freezing recovery plasmalemmae of sperms and acrosome structure, shown good sperm freezing protective effect and antifreezing effect, and then improved the fertilization rate that freezes essence.
The present invention will be further described by the following examples, and the embodiment content comprises but do not limit the scope of embodiment.
Embodiment
Embodiment 1:
Weighing 0.2g Tris, 1.2g TES, 2g lactose, 2g glucose, 0.2g raffinose, 10000IU penicillin, 5mg streptomycin sulphate are dissolved in the 60mLMilli-Q ultra-pure water successively, add the 20mL egg yolk again, mix Hou Jiashui and are settled to 100mL; In the speed of 7000g centrifugal 1 hour, collect supernatant and make semen diluent; The glycerine that adds 10% (volume ratio) in semen diluent is made the anti-icing fluid that semen freezing is preserved; (22-25 ℃) at ambient temperature mixes the macaque seminal fluid by 1: 9 volume ratio with dilution in the test tube of 4mL, test tube is sealed put into the beaker that the 200mL room temperature water is housed then, and this beaker is put into 4 ℃ the slow cooling balance 2 hours of refrigerator; In test tube, add isopyknic anti-icing fluid that contains glycerine that is chilled to 4 ℃ in advance, mix, will dilute the seminal fluid branch freezing straw of 0.25mL of packing into and put into the liquid nitrogen preservation.
Macaque seminal fluid to freezing preservation, revived motion, time-to-live, the plasmalemmae of sperms percentage of head rice of sperm after the arachidonic acid FITC-PNA fluorescence colour measuring frozen sperm that connects with living body fluorescent dyestuff propidium iodide PI decoration method and fluorescein isothiocyanate is recovered respectively, the percentage of head rice of perforatorium, its result shows: the revived motion of freezing recovery sperm is 56%, in 37 ℃ cultivate 12 hours after the sperm motility degree still can maintain more than 30%, the plasmalemmae of sperms percentage of head rice is 57%, and acrosomal integrity is 82%.It is in vitro fertilization to utilize freezing recovery sperm and macaque egg mother cell to carry out, and fertilization rate is 82%, and fertilized egg is 39% at ectogenesis to the developmental rate of blastaea.The blastaea of producing is through implantation and produce filial generation smoothly after the embryo transplantation.
Embodiment 2:
Weighing 0.2g Tris, 1.2g TES, 2g lactose, 2g glucose, 0.2g raffinose, 10000IU penicillin, 5mg streptomycin sulphate are dissolved in the 60mLMilli-Q ultra-pure water successively, add the 20mL egg yolk again, mix Hou Jiashui and are settled to 100mL., collect supernatant and make semen diluent after centrifugal 1 hour in the speed of 7000g.The glycerine that adds 10% (volume ratio) in semen diluent is made the anti-icing fluid that semen freezing is preserved.(22-25 ℃) at ambient temperature mixes the seminal fluid of Assamese macaque by 1: 9 volume ratio with dilution in the test tube of 4mL.Put into the beaker that the 200mL room temperature water is housed after then test tube being sealed, and this beaker is put into 4 ℃ the slow cooling balance 2 hours of refrigerator.In test tube, divide and add isopyknic anti-icing fluid that contains glycerine that is chilled to 4 ℃ in advance five times, each 1/5 volume that adds, each 5 minutes at interval, the final concentration of glycerine is 5%, after mixing, packing dilution seminal fluid is gone in the freezing straw of 0.25mL, places liquid nitrogen surface top 5cm to sentence 30 ℃/min after freezing 10 minutes straw, puts into liquid nitrogen and preserves.
To freezing preservation Assamese macaque seminal fluid, revived motion, time-to-live, plasmalemmae of sperms percentage of head rice with the measuring frozen sperm recovery back sperm identical with embodiment 1, the percentage of head rice of perforatorium, its result shows: the revived motion of freezing recovery sperm can reach 61%, in 37 ℃ cultivate 12 hours after the sperm motility degree still maintain more than 20%, the plasmalemmae of sperms percentage of head rice is 64%, and acrosomal integrity is 85%.It is in vitro fertilization to utilize freezing recovery sperm to carry out, and fertilization rate can reach 53%.
Embodiment 3:
Weighing 0.2g Tris, 1.2g TES, 2g lactose, 2g glucose, 0.2g raffinose, 10000IU penicillin, 5mg streptomycin sulphate are dissolved in the 60mLMilli-Q ultra-pure water successively, add the 20mL egg yolk again, mix Hou Jiashui and are settled to 100mL; In the speed of 7000g centrifugal 1 hour, collect supernatant and make semen diluent.The glycerine that adds 10% (volume ratio) in semen diluent is made the anti-icing fluid that semen freezing is preserved; (22-25 ℃) at ambient temperature, the seminal fluid of machin is mixed in the test tube of 4mL by 1: 9 volume ratio with dilution, put into the beaker that the 200mL room temperature water is housed after then test tube being sealed, and this beaker put into 4 ℃ the slow cooling balance 2 hours of refrigerator, in test tube, divide and add isopyknic anti-icing fluid that contains glycerine that is chilled to 4 ℃ in advance five times, each 1/5 volume that adds, each 5 minutes at interval, the final concentration of glycerine is 5%, after mixing, to dilute the seminal fluid branch and pack into and place in the freezing straw of 0.25mL liquid nitrogen surface top 5cm to sentence 30 ℃/min after freezing 10 minutes, and put into liquid nitrogen and preserve.
Seminal fluid to freezing preservation machin, revived motion, time-to-live, plasmalemmae of sperms percentage of head rice with the measuring frozen sperm recovery back sperm identical with embodiment 1, the percentage of head rice of perforatorium, its result shows, the revived motion of freezing recovery sperm is 50%, the plasmalemmae of sperms percentage of head rice is 59%, and acrosomal integrity is 83%.
Claims (8)
1. ultralow temperature cryopreservation method of macaca primate semen comprises the steps:
(1) get 0.2gTr i s, 1.2g TES, 2g lactose, 2g glucose, 0.2g raffinose, 10000IU penicillin G sodium salt, the 5mg streptomycin sulphate is dissolved in the 60mL Mil li-Q ultra-pure water successively, add the 20mL egg yolk, mix Hou Jiashui and be settled to 100mL;
(2) with solution centrifugal 1 hour, collect supernatant and make dilution with 7000g;
(3) glycerine of getting 45mL dilution adding 5mL is made anti-icing fluid, is chilled to 4 ℃ in advance;
(4) at room temperature, seminal fluid is mixed in test tube, seals by 1: 9 volume ratio with dilution,, put into the slow cooling of 4 ℃ of refrigerators balance again 2 hours with room-temperature water bath;
(5) in test tube, add isopyknic anti-icing fluid, divide in the straw of packing into after mixing and in liquid nitrogen, preserve; Divide in the 0.25mL straw of packing into after mixing and in liquid nitrogen, preserve;
(6) freezing straw was thawed 2 minutes by being transferred to rapidly in the liquid nitrogen in 37 ℃ the water-bath during recovery.
2. freezing and storing method according to claim 1, the room temperature that it is characterized in that described method step (4) is 22-25 ℃.
3. according to claim 1,2 described freezing and storing methods, it is characterized in that described method step (5) is to divide anti-icing fluid five times to add test tube, add 1/5 volume at every turn, each 5 minutes at interval, the final concentration of glycerine was 5%.
4. according to claim 1,2 described freezing and storing methods, it is characterized in that described method step (5) is that straw was placed 5cm place, liquid nitrogen surface top freezing 10 minutes, rate of temperature fall is 30 ℃/min, preserves in liquid nitrogen again.
5. freezing and storing method according to claim 3 is characterized in that described method step (5) is that straw was placed 5cm place, liquid nitrogen surface top freezing 10 minutes, and rate of temperature fall is 30 ℃/min, preserves in liquid nitrogen again.
6. according to claim 1,2 described freezing and storing methods, it is characterized in that described method step (5) straw volume is 0.25mL.
7. freezing and storing method according to claim 3 is characterized in that described method step (5) straw volume is 0.25mL.
8. freezing and storing method according to claim 4 is characterized in that described method step (5) straw volume is 0.25mL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102578074A (en) * | 2011-12-22 | 2012-07-18 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for preparing livestock sex control frozen sperm by adding antioxidants of CAT (Catalase) and VE (Vitamin E) |
| CN104521944A (en) * | 2014-12-31 | 2015-04-22 | 云南农业大学 | Animal-origin-free antifreezing solution and cryopreservation method thereof on seminal liquid of machin |
| CN104521945A (en) * | 2014-12-31 | 2015-04-22 | 云南农业大学 | Glycerin-free ultra deep cryopreservation method for primate seminal fluid |
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| CN1559440A (en) * | 2004-03-12 | 2005-01-05 | 中国科学院昆明动物研究所 | Low-temperature cryopreservation method for semen of gayals |
| CN1775020A (en) * | 2005-12-15 | 2006-05-24 | 上海交通大学 | Dog-like sperm super-low temperature freezing preservation antifreeze solution |
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2010
- 2010-06-12 CN CN201010198716A patent/CN101843238A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1559440A (en) * | 2004-03-12 | 2005-01-05 | 中国科学院昆明动物研究所 | Low-temperature cryopreservation method for semen of gayals |
| CN1775020A (en) * | 2005-12-15 | 2006-05-24 | 上海交通大学 | Dog-like sperm super-low temperature freezing preservation antifreeze solution |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102578074A (en) * | 2011-12-22 | 2012-07-18 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for preparing livestock sex control frozen sperm by adding antioxidants of CAT (Catalase) and VE (Vitamin E) |
| CN104521944A (en) * | 2014-12-31 | 2015-04-22 | 云南农业大学 | Animal-origin-free antifreezing solution and cryopreservation method thereof on seminal liquid of machin |
| CN104521945A (en) * | 2014-12-31 | 2015-04-22 | 云南农业大学 | Glycerin-free ultra deep cryopreservation method for primate seminal fluid |
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Application publication date: 20100929 |