CN101342192A - Quick freezing conservation method for bastard halibut spermatozoon - Google Patents
Quick freezing conservation method for bastard halibut spermatozoon Download PDFInfo
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- CN101342192A CN101342192A CNA2007100436114A CN200710043611A CN101342192A CN 101342192 A CN101342192 A CN 101342192A CN A2007100436114 A CNA2007100436114 A CN A2007100436114A CN 200710043611 A CN200710043611 A CN 200710043611A CN 101342192 A CN101342192 A CN 101342192A
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- bastard halibut
- spermatozoon
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Abstract
A bastard halibut sperm rapid cryopreservation method relates to a cryopreservation method for marine fish sperm and provides a bastard halibut sperm cryopreservation method with simple operation, rapid preservation and high sperm living rate. The present invention contains diluent, antifreeze agent and the confection of hormone cryoprotective liquid and is preserved in liquid nitrogen. The present invention is characterized in that the diluted natural seawater is used as the diluent for preserving sperms which can not activate sperms; glycol with 14g/100ml density is adopted as the anti-freeze agent; the diluent and the antifreeze agent are used as the low-temperature hormone cryoprotective liquid for bastard halibut sperms and the low-temperature hormone cryoprotective liquid is precooled in a 4 DEG C thermostatic box; the sperm and the hormone cryoprotective liquid are evenly mixed in an eccentric pipe according to the proportion of 1:1 to 1:3; the eccentric pipe is placed below the port of a liquid nitrogen jar for balancing under -20 DEG C for 1 minutes and then rapidly thrown into the liquid nitrogen for preservation. The present invention is used for bastard halibut sperm rapid cryopreservation.
Description
Technical field:
The present invention relates to seawater fish sperm store method.
Background technology
Brown Paralichthys olivaceus (Paralichthys olivacens) is commonly called as flatfish, belongs to Pleuronectiformes, and Bothidae, Paralichthys are the warm warm nature demersal fishes in coastal waters.Its NATURAL DISTRIBUTION district mainly is coastal area of china, Korea, Japan and sea area, Muscovite Asia.Its adaptability is strong, and growth is fast, individual greatly, fine and tender taste is delicious, and effects such as eliminating inflammation and expelling toxin, invigorating the spleen and benefiting QI are arranged, and is one of famous and precious important sea-farming kind.Because the international market wilderness demand, from the end of the eighties in last century to the beginning of the nineties, brown Paralichthys olivaceus suffers a large amount of fishing for, resource is subjected to serious destruction.For the deficiency that remedies resource with satisfy the demand in market, at the beginning of the nineties in last century, some kinds of Europe such as turbot that China introduces have become one of principal item of batch production sea-farming.And brown Paralichthys olivaceus is China Tu kind, fishes for influence with factor such as environment owing to be subjected to transition, and germ plasm resource is fewer and feweri.
The freezing and storing method of existing turbot, halibut spermatozoon, this method is: the diluent of sperm is artificial fully preparation such as MPRS, TS-19, TS-2; Antifreeze is the dimethyl sulfoxine (DMSO) of 10% concentration; The 6cm place is 10 minutes on the cool-down method liquid nitrogen surface, liquid nitrogen surface last 5 minute, enters liquid nitrogen and preserves.Sperm motility rate is the highest by 70%.
The freezing preservation relation of bastard halibut spermatozoon is fit to the method that the bastard halibut spermatozoon quick freezing is preserved to this species conservation of China but also lack at present.Prior art is at first measured refining osmotic pressure (mOsm/kg) with the freezing point osmotic pressure instrument, carry out the preparation of spermatozoa diluent according to osmotic pressure, spermatozoa diluent mainly adopts preparations such as NaCl, KCl, glucose, and make the diluent that is suitable for the sperm preservation need could obtain through the experiment of repeated multiple times; The antifreeze dimethyl sulfoxine, dimethyl sulfoxine is bigger to the toxic action of sperm, influences the preservation effect of sperm; The general application program cooling instrument cooling of temperature-fall period needs to be equipped with the programmed cooling instrument; The preservation container is a liquid nitrogen container.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of easy and simple to handle, are fit to the rapid saving bastard halibut spermatozoon, can obtain the freezing and storing method of higher sperm motility rate simultaneously.
Technical scheme of the present invention comprises diluent, antifreeze, frozen solution preparation, sperm mixes with protection liquid to be put into centrifuge tube input liquid nitrogen and preserves, it is characterized in that natural sea-water is diluted, with the sea-water activated sperm of dilution, with the dilution sea water that can not activate sperm as the diluent of preserving sperm; Adopting concentration is 14g/100ml ethylene glycol, or concentration is that 3g/100ml ethylene glycol+concentration is that the 3g/100ml dimethyl sulfoxine is as antifreeze; Above-mentioned diluent+antifreeze is as frozen solution, with the calorstat pre-cooling of frozen solution at 4 ℃; Sperm and frozen solution are put into centrifuge tube in 1: 1 or 1: 1.5 or 1: 2 or 1: 2.5 or 1: 3 ratio uniform mixing, and centrifuge tube is placed-20 ℃ of balance 1min under the liquid nitrogen container mouth, drop in the liquid nitrogen fast then and preserve.
The present invention is with the diluent of the natural sea-water of brown Paralichthys olivaceus living environment dilution as bastard halibut spermatozoon, avoiding manually preparing spermatozoa diluent will be through repeatedly testing the loaded down with trivial details operation that could determine various ion components and ion concentration repeatedly, the diluent of artificial preparation is owing to lack some natural trace element simultaneously, so the diluent of artificial preparation can not satisfy the needs of preserving sperm fully, preservation effect to sperm is bad, influences the survival rate of sperm.It is dimethyl sulfoxine that the prior art sperm is preserved the antifreeze that adopts; and spent glycol of the present invention (EG) adds in the diluent as freeze proof protection liquid as antifreeze; preserve bastard halibut spermatozoon; the freezing survival rate of preserving the back acquisition of bastard halibut spermatozoon is reached more than 90%, become live time to reach 255s ± 13.45s.Adopt EG (3ml/100ml)+two kinds of antifreezes of DMSO (3ml/100ml) as the combination antifreeze, except that can obtaining freezing preferably preservation effect (survival rate of freezing back sperm reaches more than 90%), two kinds of combination antifreezes can also prolong the one-tenth live time of freezing back sperm, survive for up to 540s ± 60s, than the long 285s of one-tenth live time of single spent glycol preservation.The present invention drops into liquid nitrogen fast and preserves by-20 ℃ of balances under the liquid nitrogen container mouth 1 minute, and step is simple, does not need expensive programmed cooling instrument, reaches the better preserve effect equally.Only need wear liquid nitrogen container and a thermos flask that 5-10 rises during field operation, just can carry out the freezing preservation of sperm, avoided present field acquisition to sperm must take back laboratory and just can carry out freezing preservation, long or the long-distance transport of sperm acquisition time can cause very big influence to the freezing preservation of sperm, directly has influence on the survival rate of frozen sperm.
The specific embodiment
The present invention adopts natural sea-water (its salinity the is 31) dilution of brown Paralichthys olivaceus living environment, it for example is 0.5,1,1.5,2,2.5,3 times of 31 sea water dilution with salinity, sea-water activated sperm with dilution, with the dilution sea water that can not activate sperm as the diluent of preserving sperm, after freezing is preserved, the diluent that the survival rate that detects sperm is determined the most suitable freezing preservation dilutes the diluent that 1 times of sea water is suitable as bastard halibut spermatozoon most through experimental selection; By different antifreezes, variable concentrations is to the comparison of sperm freezing preservation effect, determine that ethylene glycol (EG) is as antifreeze, concentration is 14g/100ml, or EG (3g/100ml) adds DMSO (3g/100ml) and adds in the diluent freezing protection liquid as brown Paralichthys olivaceus to as antifreeze, frozen solution is placed 4 ℃ of following pre-coolings, the protection liquid of pre-cooling is taken out, sperm can be by 1: 1 with protection liquid, 1.5,2,2.5,3 dilution proportion, effect by 1: 15 dilution proportion is best, the sperm that dilution is good is packed in the plastic centrifuge tube of 200 μ l, placed under the liquid nitrogen container mouth-20 ℃ of balances 1 minute, and then it was dropped into liquid nitrogen fast and preserve; Centrifuge tube proposes from liquid nitrogen fast when thawing, and places 38 ℃ of water-baths to thaw.Adopt the freezing preservation bastard halibut spermatozoon of this method, the motility rate of freezing back sperm reaches more than 90%.This method compares with the method for preserving turbot, Paralichthys olivaceus, and the motility rate of freezing back sperm improves more than 20%, is that present fresh water and seawater fish sperm freezing are preserved the highest method of back acquisition sperm motility rate.
Claims (3)
1. quick freezing conservation method for bastard halibut spermatozoon, comprise diluent, antifreeze, frozen solution preparation, sperm mixes with protection liquid to be put into centrifuge tube input liquid nitrogen and preserves, it is characterized in that natural sea-water is diluted, with the sea-water activated sperm of dilution, with the dilution sea water that can not activate sperm as the diluent of preserving sperm; Adopting concentration is that 14g/100ml ethylene glycol is as antifreeze; Above-mentioned diluent+antifreeze is as frozen solution, with the calorstat pre-cooling of frozen solution at 4 ℃; Sperm and frozen solution are put into centrifuge tube in 1: 1 or 1: 1.5 or 1: 2 or 1: 2.5 or 1: 3 ratio uniform mixing, and centrifuge tube is placed-20 ℃ of balance 1min under the liquid nitrogen container mouth, drop in the liquid nitrogen fast then and preserve.
2. quick freezing conservation method for bastard halibut spermatozoon according to claim 1, it is characterized in that adopting concentration is that 3g/100ml ethylene glycol+concentration is 14g/100ml ethylene glycol antifreeze for the 3g/100ml dimethyl sulfoxine substitutes concentration.
3. quick freezing conservation method for bastard halibut spermatozoon according to claim 1 and 2 is characterized in that adopting the diluent of 1 times of sea water of dilution as bastard halibut spermatozoon.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100436114A CN101342192B (en) | 2007-07-09 | 2007-07-09 | Quick freezing conservation method for bastard halibut spermatozoon |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007100436114A CN101342192B (en) | 2007-07-09 | 2007-07-09 | Quick freezing conservation method for bastard halibut spermatozoon |
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| CN101342192A true CN101342192A (en) | 2009-01-14 |
| CN101342192B CN101342192B (en) | 2011-05-18 |
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| CN2007100436114A Expired - Fee Related CN101342192B (en) | 2007-07-09 | 2007-07-09 | Quick freezing conservation method for bastard halibut spermatozoon |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524241A (en) * | 2010-12-16 | 2012-07-04 | 宁波大学 | Additive for freezing storage protective liquid of fish spermatid |
| CN103348932A (en) * | 2013-05-31 | 2013-10-16 | 中国科学院海洋研究所 | Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus |
| WO2018058541A1 (en) * | 2016-09-30 | 2018-04-05 | 台北荣民总医院 | Anti-freezing agent for protecting biological tissue during freezing treatment and preparation method therefor |
| CN112741078A (en) * | 2020-12-24 | 2021-05-04 | 山东省海洋生物研究院 | Hexagrammos otakii sperm productive cryopreservation method |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2007
- 2007-07-09 CN CN2007100436114A patent/CN101342192B/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102524241A (en) * | 2010-12-16 | 2012-07-04 | 宁波大学 | Additive for freezing storage protective liquid of fish spermatid |
| CN103348932A (en) * | 2013-05-31 | 2013-10-16 | 中国科学院海洋研究所 | Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus |
| CN103348932B (en) * | 2013-05-31 | 2015-05-20 | 中国科学院海洋研究所 | Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus |
| WO2018058541A1 (en) * | 2016-09-30 | 2018-04-05 | 台北荣民总医院 | Anti-freezing agent for protecting biological tissue during freezing treatment and preparation method therefor |
| US11160271B2 (en) * | 2016-09-30 | 2021-11-02 | Taipei Veterans General Hospital | Anti-freezing agent for protecting biological tissue during freezing treatment and preparation method therefor |
| US20210360916A1 (en) * | 2016-09-30 | 2021-11-25 | Taipei Veterans General Hospital | Anti-freezing agent for protecting biological tissue during freezing treatment and preparation method therefor |
| US11596145B2 (en) * | 2016-09-30 | 2023-03-07 | Taipei Veterans General Hospital | Anti-freezing agent for protecting biological tissue during freezing treatment and preparation method therefor |
| CN112741078A (en) * | 2020-12-24 | 2021-05-04 | 山东省海洋生物研究院 | Hexagrammos otakii sperm productive cryopreservation method |
| CN112741078B (en) * | 2020-12-24 | 2022-03-25 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | Hexagrammos otakii sperm productive cryopreservation method |
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| CN101342192B (en) | 2011-05-18 |
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