CN104304234A - Freezing protection solution and preservation method of Plectropomus leopardus sperms - Google Patents
Freezing protection solution and preservation method of Plectropomus leopardus sperms Download PDFInfo
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- CN104304234A CN104304234A CN201410473950.6A CN201410473950A CN104304234A CN 104304234 A CN104304234 A CN 104304234A CN 201410473950 A CN201410473950 A CN 201410473950A CN 104304234 A CN104304234 A CN 104304234A
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- freezing
- sperms
- sperm
- liquid nitrogen
- protection solution
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Abstract
The invention discloses a freezing protection solution of Plectropomus leopardus sperms. The freezing protection solution comprises 34.23g of mycose, 0.9L of fetal calf serum, and water added to the total volume of 1L; and the pH value and the osmotic pressure of the freezing protection solution are 7.45 and 324mOsmol/Kg respectively. The preservation method comprises the following steps: carrying out isopyknic mixing on a seminal fluid and the freezing protection solution in 2ml of a freezing tube, allowing the freezing tube with the obtained mixed solution to stay in a position 5cm higher than a liquid nitrogen surface for 3min, and directly putting the freezing tube with the mixed solution into liquid nitrogen. The freezing tube with sperms frozen in the liquid nitrogen is taken out, and is processed in a 37DEG C water bath for 1min in order to unfreeze. Compared with freezing solutions in the prior art, the freezing protection solution has the advantages of very low toxicity to the sperms, and provision of nutrients needed by the sperms.
Description
technical field
The present invention relates to the freezen protective that gill sour jujube perch belongs to fish sperm, particularly to leopard line gill sour jujube perch sperm freezing protection liquid and store method thereof.
background technology
Gill sour jujube perch is famous and precious seafood fish renowned in the world, delicious meat, and nutritious, economic worth is very high.The propagating artificially of some kinds (as leopard line gill sour jujube perch) that gill sour jujube perch belongs to wherein economizes universal and popularization rapidly in China Guangdong and Hainan etc., becomes the coastal important mariculture kind in south.
Ultra-cryofreezing preservation seminal fluid is the effective way of protection fish elite germplasm, and the work of China's fish sperm freezen protective mainly concentrates on freshwater fish and the cold aqueous seawater fishs such as flounder flounder class and large yellow Crocker such as " four large Chinese carps ".The sperm physiological properties of fish not of the same race etc. are widely different, and therefore the optimum condition of Ultra-cryofreezing preservation also varies.So far, there is not yet the research report that gill sour jujube perch belongs to fish sperm Ultra-cryofreezing preservation.
Frozen solution is most crucial to sperm storage effect, mainly adopts permeability antifreeze (methyl-sulfoxide, dimethyl formamide, glycerine, methyl alcohol, ethylene glycol etc.) and salting liquid (NaCl, KCl, Na at present
2cO
3, NaH
2pO
4, Na
2hPO
4, MgSO
4, MgCl
2deng) mixed preparing.But permeability antifreeze is mostly inorganic chemicals, itself has certain toxicity to sperm; Salting liquid can not provide necessary nutriment to sperm.
The various compositions of sperm of the prior art freeze proof protection liquid all may have certain toxicity to sperm, and formula is simplified by the present invention, is conducive to alleviating the harmful effect of freeze proof protection liquid to sperm itself.Trehalose is a kind of antifreeze of impermeability, sperm dehydration can be made to reduce in refrigerating process and produce ice crystal damage, thus play the effect of protection sperm.Trehalose is macromolecular substances, and can not enter into sperm inside through cell membrane, relative to permeability antifreeze, the toxicity of trehalose is lower.Hyclone is one of important culture fluid of cell chulture, containing abundant amino acid and vitamin, for sperm provides abundant nutriment.
summary of the invention
The object of the present invention is to provide a kind of leopard line gill sour jujube perch sperm freezing to protect liquid, solve the toxicity of the permeability antifreeze mixing salt solution existence adopted at present and lack the problems such as nutrition.
Another object of the present invention is to provide a kind of above-mentioned leopard line gill sour jujube perch sperm cryopreservation method, makes up current gill sour jujube perch and belongs to the deficiency that fish there is no relevant Techniques of preserving.
The technical scheme of technical solution problem of the present invention: a kind of leopard line gill sour jujube perch sperm freezing protection liquid, formulated by following component: trehalose 34.23 grams, hyclone 0.9 liter, ultra-pure water is settled to 1 liter; Described frozen solution PH is 7.45, and osmotic pressure is 324mOsmol/Kg.
Leopard line gill sour jujube perch sperm cryopreservation method of the present invention, mixed with described frozen solution equal-volume by seminal fluid in 2ml cryopreservation tube, then on liquid nitrogen surface, the stop of 5cm place was preserved in direct plunge into Liquid Nitrogen after 3 minutes; When thawing, the cryopreservation tube having sperm will be chilled in liquid nitrogen and take out, in 37 DEG C of water-baths 1 minute.
The present invention compared with prior art, has following advantage: very low to the toxicity of sperm, and can provide sperm desired nutritional material.
Embodiment
Embodiment 1
1. the preparation of frozen solution: trehalose 34.23 grams, hyclone 0.9 liter, ultra-pure water is settled to 1 liter (PH=7.45, osmotic pressure=324mOsmol/Kg).
2. semen collection: with eugenol anesthesia leopard line gill sour jujube perch sexual maturity milter, dry fish body and avoid sperm and contact with sea water, light pressure belly collects seminal fluid in 2ml cryopreservation tube.
3. freezing: to be mixed with frozen solution equal-volume by seminal fluid in cryopreservation tube, then on liquid nitrogen surface, the stop of 5cm place was preserved in direct plunge into Liquid Nitrogen after 3 minutes.
4. thaw: the cryopreservation tube having sperm will be chilled in liquid nitrogen and take out, in 37 DEG C of water-baths 1 minute.
5. sperm viability detects: the vigor after utilizing counting machine assisted sperm analysis system detection leopard line gill sour jujube perch sperm to thaw is 36.33%, close to fresh smart vigor 41.69%.
The same terms is with under operation, and three kinds of permeability antifreeze methyl-sulfoxides, dimethyl formamide and glycerine preserve the vigor of sperms between 20%-30%, and preservation effect is all lower than the freeze proof protection liquid that the present invention adopts.
Claims (2)
1. leopard line gill sour jujube perch sperm freezing protection liquid, it is characterized in that: trehalose 34.23 grams, hyclone 0.9 liter, ultra-pure water is settled to 1 liter; Described frozen solution pH value is 7.45, and osmotic pressure is 324mOsmol/Kg.
2. one kind uses the leopard line gill sour jujube perch sperm cryopreservation method of frozen solution described in claim 1, it is characterized in that: mixed with described frozen solution equal-volume by seminal fluid in 2ml cryopreservation tube, then on liquid nitrogen surface, the stop of 5cm place was preserved in direct plunge into Liquid Nitrogen after 3 minutes; When thawing, the cryopreservation tube having sperm will be chilled in liquid nitrogen and take out, in 37 DEG C of water-baths 1 minute.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410473950.6A CN104304234B (en) | 2014-09-17 | 2014-09-17 | Leopard line gill sour jujube perch sperm freezing protection liquid and store method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410473950.6A CN104304234B (en) | 2014-09-17 | 2014-09-17 | Leopard line gill sour jujube perch sperm freezing protection liquid and store method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104304234A true CN104304234A (en) | 2015-01-28 |
| CN104304234B CN104304234B (en) | 2016-05-18 |
Family
ID=52359675
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| CN201410473950.6A Active CN104304234B (en) | 2014-09-17 | 2014-09-17 | Leopard line gill sour jujube perch sperm freezing protection liquid and store method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112352777A (en) * | 2020-11-19 | 2021-02-12 | 中山大学 | Sperm cryopreservation liquid and application thereof in sperm cryopreservation of Bostrichthys sinensis |
| CN114467920A (en) * | 2022-01-28 | 2022-05-13 | 海南大学 | Low-temperature preservation diluent, preservation method and application of plectropomus leopardus semen |
| CN116998479A (en) * | 2023-10-07 | 2023-11-07 | 海南大学三亚南繁研究院 | Ultralow-temperature preservation solution, method for preserving semen of Perch gill and application of ultralow-temperature preservation solution |
Citations (3)
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| WO2001028326A1 (en) * | 1999-10-20 | 2001-04-26 | Istituto Sperimentale Italiano 'lazzaro Spallanzani' | Method for cryopreservation of teleostei semen |
| CN102726369A (en) * | 2012-06-18 | 2012-10-17 | 中国水产科学研究院黄海水产研究所 | Ultralow temperature cryopreservation application method of Epinephelus oanceolutus sperms |
| CN103314949A (en) * | 2013-05-31 | 2013-09-25 | 中国科学院海洋研究所 | Ultralow temperature cryopreservation method for preserving sperm of Pacific cod |
-
2014
- 2014-09-17 CN CN201410473950.6A patent/CN104304234B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001028326A1 (en) * | 1999-10-20 | 2001-04-26 | Istituto Sperimentale Italiano 'lazzaro Spallanzani' | Method for cryopreservation of teleostei semen |
| CN102726369A (en) * | 2012-06-18 | 2012-10-17 | 中国水产科学研究院黄海水产研究所 | Ultralow temperature cryopreservation application method of Epinephelus oanceolutus sperms |
| CN103314949A (en) * | 2013-05-31 | 2013-09-25 | 中国科学院海洋研究所 | Ultralow temperature cryopreservation method for preserving sperm of Pacific cod |
Non-Patent Citations (3)
| Title |
|---|
| KADOO MIYAKI等: "Cryopreservation of kelp grouper Epinephelus moara sperm using only a trehalose solution", 《FISHERIES SCIENCE》, 31 December 2005 (2005-12-31), pages 457 - 458 * |
| 昝林森等: "海藻糖和牛血清白蛋白对牛精液的冷冻效果", 《西北农林科技大学学报(自然科学版)》, vol. 36, no. 10, 31 October 2008 (2008-10-31), pages 53 - 58 * |
| 郑乃中等: "斑马鱼精子冻存与复苏实验方法与流程", 《遗传》, vol. 39, no. 9, 30 September 2012 (2012-09-30), pages 1211 - 1216 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112352777A (en) * | 2020-11-19 | 2021-02-12 | 中山大学 | Sperm cryopreservation liquid and application thereof in sperm cryopreservation of Bostrichthys sinensis |
| CN114467920A (en) * | 2022-01-28 | 2022-05-13 | 海南大学 | Low-temperature preservation diluent, preservation method and application of plectropomus leopardus semen |
| CN114467920B (en) * | 2022-01-28 | 2022-11-29 | 海南大学 | Low-temperature preservation diluent, preservation method and application of plectropomus leopardus semen |
| CN116998479A (en) * | 2023-10-07 | 2023-11-07 | 海南大学三亚南繁研究院 | Ultralow-temperature preservation solution, method for preserving semen of Perch gill and application of ultralow-temperature preservation solution |
| CN116998479B (en) * | 2023-10-07 | 2023-12-26 | 海南大学三亚南繁研究院 | Ultralow-temperature preservation solution, method for preserving semen of Perch gill and application of ultralow-temperature preservation solution |
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| Publication number | Publication date |
|---|---|
| CN104304234B (en) | 2016-05-18 |
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