CN1309301C - Ultra low temperature refrigeration method for preserving sperm of sturgeon - Google Patents
Ultra low temperature refrigeration method for preserving sperm of sturgeon Download PDFInfo
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- CN1309301C CN1309301C CNB031512631A CN03151263A CN1309301C CN 1309301 C CN1309301 C CN 1309301C CN B031512631 A CNB031512631 A CN B031512631A CN 03151263 A CN03151263 A CN 03151263A CN 1309301 C CN1309301 C CN 1309301C
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Abstract
The present invention relates to a preservation method by freezing sperms of acipenseridae fish at ultralow temperature, which relates to a preservation method for fish sperms. The present invention aims to provide a method for preserving sperms of acipenseridae fish with a higher survival rate. In the preservation method, a freezing-point osmometer, a low-temperature creature cooling device and a liquid nitrogen container are used. The present invention is characterized in that the osmotic pressure of sperms of acipenseridae fish is measured, preservation liquid is prepared according to the osmotic pressure, and freeze-resistant protection agents and the concentration are determined; the accurate temperature of the liquid nitrogen container is measured through the low-temperature creature cooling device, an optimal cooling temperature layer is established, and diluting liquid with DMSO is used as freezing protection liquid; the sperms and the protection liquid are mixed according to the proportion of 3:1 for diluting the sperms, and the sperms are equalized; when the osmotic pressure of the sperms is most suitable for preserving the sperms, the sperms are filled in a centrifugal tube cooled to minus 5 DEG C through the low-temperature creature cooling device and equalized for 10 minutes, and the sperms are put in the vapor of liquid nitrogen at a temperature of minus 160 to minus 180 DEG C and equalized for 20 minutes and then preserved in the liquid nitrogen; when defreezed, the sperms are heated to a temperature of minus 160 to minus 180 DEG C from a temperature of minus 196 DEG C and equalized for 20 minutes, and then the sperms are defreezed in a water bath at a temperature of 38 to 40 DEG C. The present invention is used for aquiculture.
Description
Technical field:
The present invention relates to the method for the freezing preservation of fish sperm.
Background technology
Sturgeon section fish are rare precious water biological species, and sturgeon section fish sperm preservation relation still, also lacks the successful methods that can be used for preserving sturgeon section fish sperm at present to the protection of rare species.
Prior art is preserved silver carp, bighead, grass carp, carp freshwater fish sperm with liquid nitrogen container, liquid nitrogen temperature-196 ℃, in the preservation process, by the manual temperature of measuring in the liquid nitrogen container, because of measure error, cause the preservation effect instability, simultaneously easily, the technical best osmotic pressure value of not measuring the sperm freezing preservation of freezing preservation, the frozen sperm motility rate is not high.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of sturgeon section fish sperm of preserving, and have the freezing and storing method of higher motility rate simultaneously.
Technical scheme adapted freezing-point osmometer of the present invention, low-temperature biological cooling instrument and liquid nitrogen container; By the indirect determination of NaCl solution and the direct mensuration of freezing-point osmometer, at first measure the osmotic pressure value of sturgeon section fish sperm itself; Carry out the preparation that sperm is preserved formula of liquid by osmotic pressure value, sperm is preserved liquid and is adopted NaCl, KCl, glucose preparation, and the concentration that the concentration of NaCl is 0.3～0.75%, the concentration of KCl is 0.02-0.1%, glucose is 1-1.6%; Determine freezeproof protectant kind and the concentration that sturgeon section fish sperm is preserved then, adopt DMSO as antifreeze, concentration is 10～14%; Directly measure the accurate temperature of liquid nitrogen container by the low-temperature biological cooling instrument, establish best cooling temperature layer; As sturgeon section fish sperm cryogenic freezing protection liquid, sperm dilutes by 3: 1 with the ratio of protection liquid with dilution+DMSO antifreeze; Balance Treatment 2～3h under 2～4 ℃ low temperature, when the osmotic pressure of sperm is brought up to the suitableeest save value 800mOsm/kg, with pack into the plastic centrifuge tube of 2ml of sperm, by the low-temperature biological cooling instrument, be cooled to-5 ℃ of balance 10min, drop into-160～-180 ℃ of liquid nitrogen steam Zhong Ping Red 20min then, put into-196 ℃ of liquid nitrogen again and preserve; When thawing, the sperm of preserving is risen to-160～-180 ℃ of warm area Ping Red from-196 ℃ handle 20min, and then in 38～40 ℃ of water-baths, thaw.
The present invention directly measures the accurate temperature of liquid nitrogen container by the low-temperature biological cooling instrument, avoid error, establish the cooling temperature layer of-5 ℃ →-180 ℃ → liquid nitrogen the best, reach stable preservation effect, and the indirect determination of employing NaCl solution and the direct mensuration of freezing-point osmometer, measure the osmotic pressure value of sperm itself, carry out the preparation that sperm is preserved liquid by osmotic pressure value.Because the present invention preserves the preparation of liquid all produces favourable sperm viability to preservation effect influence from the establishment of temperature measuring accurately and best cooling layer to sperm, not only sturgeon section fish sperm freezing sperm motility rate reaches the technique effect more than 70%, also can be used for the freezing preservation of conventional fish sperm.
Embodiment
The present invention is equipped with the FM-8P freezing-point osmometer, survey the osmotic pressure of sturgeon section fish sperm, for example measure the osmotic pressure 54～56mOsm/kg of acipenser schrencki section fish sperm, carry out the preparation that sperm is preserved liquid by osmotic pressure value, sperm is preserved liquid with NaCl, KCl, glucose preparation, the concentration that can adopt NaCl is 0.3%, the concentration of KCl is 0.03%, the concentration of glucose is the preservation liquid of 1.2% preparation, or contains the preservation liquid of NaCl0.5g, KCl0.03g, glucose 1.2g in the 100ml solution; Adopt DMSO as antifreeze, concentration is 12%; Sperm dilutes by 3: 1 with the ratio of protection liquid; Behind Balance Treatment 2～3h under 2～4 ℃ the low temperature,, lower the temperature by the low-temperature biological cooling instrument with pack into the plastic centrifuge tube of 2ml of sperm; Reduce to-5 ℃ of balance 10min, drop into balance 20min in-160 ℃ of liquid nitrogen steam then, put into-196 ℃ of liquid nitrogen again and preserve; When thawing, the sperm preserved is risen to-160 ℃ of warm area Balance Treatment 20min from-196 ℃, and then in 38 ℃ of water-baths, thaw.Adopt this method fish sperm motility rate to reach more than 70%.
Claims (1)
- Sturgeon section fish sperm super-low temperature freezing store method is equipped with freezing-point osmometer, low-temperature biological cooling instrument and liquid nitrogen container; It is characterized in that at first measuring the osmotic pressure of sturgeon section fish sperm by the indirect determination of NaCl solution and the direct mensuration of freezing-point osmometer; Carry out the preparation that sperm is preserved liquid by the osmotic pressure value of measuring sperm itself, sperm is preserved liquid with NaCl, KCl, glucose preparation, and the concentration of NaCl is that the concentration of 0.5g/100ml, KCl is that the concentration of 0.03g/100ml, glucose is 1.2g/100ml; Determine freezeproof protectant kind and the concentration that sturgeon section fish sperm is preserved then, adopt DMSO as antifreeze, concentration is 10～14%; Directly measure the accurate temperature of liquid nitrogen container by the low-temperature biological cooling instrument, establish the cooling temperature layer of-5 ℃ →-180 ℃ of liquid nitrogen the bests; As sturgeon section fish sperm cryogenic freezing protection liquid, sperm dilutes by 3: 1 with the ratio of protection liquid with dilution+DMSO antifreeze; Balance Treatment 2～3h under 2～4 ℃ low temperature, when the osmotic pressure of sperm is brought up to 800mOsm/kg, with pack into the plastic centrifuge tube of 2ml of sperm, by the low-temperature biological cooling instrument, reduce to-5 ℃ of balance 10min, drop into-160～-180 ℃ of liquid nitrogen steam Zhong Ping Red 20min then, put into liquid nitrogen again and preserve; When thawing, the sperm of preserving is risen to-160～-180 ℃ of warm area Ping Red from-196 ℃ handle 20min, and then in 38～40 ℃ of water-baths, thaw.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031512631A CN1309301C (en) | 2003-09-28 | 2003-09-28 | Ultra low temperature refrigeration method for preserving sperm of sturgeon |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB031512631A CN1309301C (en) | 2003-09-28 | 2003-09-28 | Ultra low temperature refrigeration method for preserving sperm of sturgeon |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1600097A CN1600097A (en) | 2005-03-30 |
| CN1309301C true CN1309301C (en) | 2007-04-11 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB031512631A Expired - Fee Related CN1309301C (en) | 2003-09-28 | 2003-09-28 | Ultra low temperature refrigeration method for preserving sperm of sturgeon |
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| CN (1) | CN1309301C (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101502256B (en) * | 2009-03-11 | 2012-02-15 | 宁波大学 | Ultra low temperature cryopreservation method for sperm of large yellow crocker and cryopreservation device |
| CN102986652B (en) * | 2012-12-25 | 2013-12-18 | 中国水产科学研究院黑龙江水产研究所 | Superfreeze preservation method of pink salmon sperm |
| CN103931606A (en) * | 2014-04-04 | 2014-07-23 | 湖南苗王生物科技有限公司 | Preparation method for vitrification freezing-thawing protection liquid of sturgeon sperms |
| CN103931607A (en) * | 2014-04-04 | 2014-07-23 | 湖南苗王生物科技有限公司 | Vitrification freezing method for sturgeon sperms |
| CN104521943A (en) * | 2014-12-09 | 2015-04-22 | 南京师范大学 | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen |
| CN107873698A (en) * | 2017-11-23 | 2018-04-06 | 广东省生物资源应用研究所 | A kind of long-term frozen store method of Amur Sturgeon sperm cryopreservation liquid and Amur Sturgeon sperm |
| CN112806353A (en) * | 2020-12-30 | 2021-05-18 | 西南大学 | Method for preserving and recovering sturgeon sperms |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU591164A1 (en) * | 1976-02-02 | 1978-02-05 | Институт Проблем Криобиологии И Криомедицины Ан Украинской Сср | Method of preserving fish sperm |
| SU786947A1 (en) * | 1979-01-04 | 1980-12-15 | Институт Проблем Криобиологии И Криомедицины Ан Украинской Сср | Method of preserving fish sperm |
| CN1342043A (en) * | 1998-10-14 | 2002-03-27 | 卡特里娜T·福雷斯特 | Method for vitrification of biological specimen |
-
2003
- 2003-09-28 CN CNB031512631A patent/CN1309301C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU591164A1 (en) * | 1976-02-02 | 1978-02-05 | Институт Проблем Криобиологии И Криомедицины Ан Украинской Сср | Method of preserving fish sperm |
| SU786947A1 (en) * | 1979-01-04 | 1980-12-15 | Институт Проблем Криобиологии И Криомедицины Ан Украинской Сср | Method of preserving fish sperm |
| CN1342043A (en) * | 1998-10-14 | 2002-03-27 | 卡特里娜T·福雷斯特 | Method for vitrification of biological specimen |
Non-Patent Citations (3)
| Title |
|---|
| 二甲亚砜对几种淡水鱼精子渗透压及成活率影响的研究 章龙珍、刘宪亭、陈松林、鲁大椿、郭峰，水生生物学报，第18卷第4期 1994 * |
| 鱼类、水生生物配子及胚胎低温冷冻保存研究进展 柳凌、刘宪亭、章龙珍、鲁大椿，淡水渔业，第27卷第3期 1997 * |
| 鲢、鲤、团头鲂和草鱼精液冷冻保存的研究 陈松林、刘宪亭、鲁大椿、章龙珍、傅朝君、方建平，动物学报，第38卷第4期 1992 * |
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| CN1600097A (en) | 2005-03-30 |
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