CN101965828B - Method for cryopreserving lutjanus argentimaculatus sperm - Google Patents
Method for cryopreserving lutjanus argentimaculatus sperm Download PDFInfo
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- CN101965828B CN101965828B CN2010102677899A CN201010267789A CN101965828B CN 101965828 B CN101965828 B CN 101965828B CN 2010102677899 A CN2010102677899 A CN 2010102677899A CN 201010267789 A CN201010267789 A CN 201010267789A CN 101965828 B CN101965828 B CN 101965828B
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Abstract
The invention relates to a method for cryopreserving and activating lutjanus argentimaculatus sperm. The method comprises the steps of collecting, diluting, cooling and freezing the lutjanus argentimaculatus sperm and is characterized in that: the cryopreservation comprises the steps of: collecting the lutjanus argentimaculatus sperm, diluting and balancing the sperm by using a diluent and an anti-freeze agent, and cryopreserving the sperm by a fragmental and gradual cooling method; and the activation method comprises the steps of: activating the sperm in the unfrozen sperm by using seawater,and performing quality detection on the sperm and comprehensive quantitative analysis on the dynamic and static characteristics of the sperm so as to accurately evaluate the sperm quality and obtain the lutjanus argentimaculatus sperm with high survival rate and strong vigor; therefore, precious wild resources and excellent biological species can be effectively stored and can be fetched at any time as necessary.
Description
Technical field
The invention belongs to the sperm freezing technical field, be specifically related to the rock salmon sperm cryopreservation method.
Background technology
The South Sea is the big epeiric sea in third place in the world, about 3,500,000 km of area
2, about 2,010,000 km of China seas under its jurisdiction area wherein
2Because its marine site is vast, water quality is good, marine resources are abundant, marine organisms kinds such as fish, shrimp, shellfish, algae, kind are numerous, and total has 4168 kinds approximately, accounts for 52.2% of national corresponding monoid kind.Along with quickening and the South Sea surrounding countries of global economy competitively develop marine economy, China also accelerates the exploitation paces of Nanhai District marine resources.But be accompanied by accelerating development of marine economy, the decline of fishery germ plasm resource has appearred in China; The maritime environment pollution situation is on the rise; Red tide is frequent; Problems such as the coastal waters deterioration of the ecological environment, these have a strong impact on and restrict the sound development of China's marine economy.Therefore, when protecting the marine eco-environment, we also will pay attention to protecting original wild resource, pay attention to the germplasm preservation, provide approach for realizing sustainable development from now on.
It is the effective way of long preservation animal germ plasm resource that sperm freezing is preserved, and all has broad application prospects at aspects such as preventing fish species extinction, fish genetic breeding and seed rearing.Lutjanidae fish and ablen and sparid class etc. be sexual conversion phenomenon all, for example grouper has " earlier female back is male ", the Le Shi jenoar has the sex reversal process of " male back is female earlier ", the about 2-5 of sex reversal required time, the variety classes difference, huge grouper reaches ability sex reversal success more than 6 years, and this has just increased the difficulty of artificial propagation.Whether fish sperm can obtain dense bright essence by the ripe sexual gland of manual compression as domestic animal so.If this method is succeedd, by adopting superfreeze to preserve the seminal fluid technology semen freezing of ripe milter is preserved so, take out when needing and carry out artificial insemination, this method not only just can solve the problem of milter deficiency, also can solve the difficult problem when female, male parent population sexual maturity is asynchronous matches.
Chinese patent discloses a kind of simple method for ultralow temperature preservation of fish sperm, application number: 200710051591.5, open (bulletin) number: CN101088511 applies for (patent right) people: Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences, address: No. 41, North Road, Jiang-Han Area, Shashi District, Jingzhou City, Hubei Province, invention (design) people: Liu Ling; Zhang Jieming; It is big to have endangered; Guo Feng; Zhu Yongjiu, summary: simple method for ultralow temperature preservation of fish sperm, it is after fish sperm is taken out, sperm and dilution were put into 4 ℃ of refrigerator inner equilibriums 20-30 minute by 1: 1～5 dilution proportion, add certain density antifreeze again, after using the packing of 0.5 milliliter of wheat tubule then, put on the bubble chamber liquid nitrogen surface thick 2.5～3 centimetres, on wide 10 centimetres of foam plates, after 20 minutes, wheat tubule on the cystosepiment is slipped in the liquid nitrogen, more all wheat tubules are changed over to temperature after 5 minutes and be interior freezing preservation in-196 ℃ the liquid nitrogen container.The present invention can be under the field condition far from the laboratory, and does not have under the situation of programmed cooling instrument or computer-controlled program cooling instrument, realizes that, superfreeze quick, easy to fish sperm preserve, and it is easy to operation, and refrigerating effect is good, and cost is low.
Summary of the invention
Purpose of the present invention aims to provide a kind of proportioning ratio of seawater fish sperm freezing liquid, improves sperm survival rate, especially at the rock salmon sperm cryopreservation method.
For achieving the above object, the present invention is achieved through the following technical solutions:
This rock salmon sperm freezing is preserved and Activiation method, comprise the collection of rock salmon sperm, dilution, cooling and freezing, it is characterized in that: described freezing preservation is that the collection of rock salmon sperm is diluted and balance sperm with dilution and antifreeze later on, adopt again segmentation progressively falling temperature method carry out freezing preservation; Described Activiation method is with the sea-water activated sperm of the seminal fluid that thaws, and carries out sperm quality at last and detects.
Above-described sperm the dilution with balance with dilution and antifreeze is that rock salmon seminal fluid and dilution and antifreeze proportioning are diluted by 1: 1～2 part by weight, and the part by weight of described dilution and antifreeze is 6%～16%.
Above-described segmentation progressively falling temperature method to carry out freezing preservation be to utilize the container that liquid nitrogen is housed, control rate of temperature fall by the control sample apart from the height of liquid nitrogen surface, temperature is by 0 ℃ of beginning, fall 10-20 ℃, segmentation is down to-80 ℃ successively at every turn, last balance 3～8 minutes, after cooling is finished, sperm is taken out from the programmed cooling instrument, put into liquid nitrogen container, in-198 ℃, preserve.
Above-described dilution is Hanks ', TS-2, TS-19 and Cortland; Described antifreeze is that weight content is the methyl-sulfoxide (DMSO) of 6%-16%.Hanks ', TS-2, TS-19 and Cortland are with KH with dilution
2PO
4, MgSO
4, NaCl, NaHCO
3, KCI, glucose be the solution of main component, the concrete composition of the Hanks ' of the art, TS-2, TS-19 and Cortland and content are with reference to Chen Songlin chief editor " fish sperm and embryo cryopreservation are preserved theory and technology " (Chinese agriculture publishing house).
It is to utilize Computer Recognition Technology and image processing techniques that above-described sperm quality detects, dynamic and static nature to sperm carries out comprehensive quantitative analysis, detect the movement locus of sperm, movement rate, distribute, sperm profile (abnormal rate), sperm quantity, viscosity, vigor etc., the difference that discloses frozen sperm and fresh spermatozoa quality directly perceived, find out dilution and the best matching method of antifreeze of freezing preservation, thereby sperm quality is made evaluation accurately, obtain the survival rate height, energetic rock salmon sperm, precious wild resource and excellent biological species mass-energy are stored enough effectively, and can when people need, take at any time.
The invention has the beneficial effects as follows:
1, adopt the dilution antifreeze to sperm dilution and balance,
Compared with prior art, method provided by the invention utilizes the existing sperm quality analytical system in laboratory (CASA) to carry out the computer-aided detection of sperm quality, by modern Computer Recognition Technology and image processing techniques, dynamic and static nature to sperm carries out comprehensive quantitative analysis, computer automation detects the movement locus of sperm, movement rate, distribute, sperm profile (abnormal rate), sperm quantity, viscosity, vigor etc., the difference that discloses frozen sperm and fresh spermatozoa quality directly perceived, find out dilution and the best matching method of antifreeze of freezing preservation, thereby sperm quality is made evaluation accurately, obtain the survival rate height, energetic rock salmon sperm, precious wild resource and excellent biological species mass-energy are stored enough effectively, and can when people need, take at any time.
Embodiment
Technical scheme of the present invention is as follows:
(1) preparation of dilution: intend filtering out suitable dilution prescription and dosage from 4 kinds of dilutions (Hanks ', TS-2, TS-19, Cortland).
(2) screening of antifreeze: filter out suitable dosage from 6%～16% permeability antifreeze methyl-sulfoxide (DMSO).
(3) selection of cool-down method: adopt progressively falling temperature method of segmentation, utilize the container that liquid nitrogen is housed, control rate of temperature fall by the control sample apart from the height of liquid nitrogen surface.
(4) sperm quality detects: utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer-aided detection of sperm quality, by modern Computer Recognition Technology and image processing techniques, sound attitude feature to sperm is carried out comprehensive quantitative analysis, the movement locus of computer automation detection sperm, movement rate, distribution, sperm profile (abnormal rate), sperm quantity, viscosity, vigor etc., intuitively disclose the difference of frozen sperm and fresh spermatozoa quality, thereby accurately estimate the effect of freezing preservation.
Embodiment 1
(1) select the rock salmon of body weight 4.5kg, the human chorionic gonadotrophin (HCG) of dorsal muscles injection 150IU/kg, during observe the variation of milter sperm volume;
(2) proportioning of antifreeze: antifreeze be Hank ' s, TS-2, TS-19 and Cortland4 kind base fluid respectively with weight content 6,8,10,12,14 and 16%DMSO carry out proportioning, it is stand-by that the liquid that proportioning is good is positioned over 4 ℃ of refrigerators, in 1: 2 ratio with seminal fluid and antifreeze proportioning;
(3) the programmed cooling instrument is lowered the temperature: open computer and liquid nitrogen pump, open the programmed cooling instrument, make cooling storehouse temperature in 15～20 minutes, be down to 0 ℃, wait for application of sample, 4 kinds of base fluids are mixed with sperm with the DMSO mixed liquor of variable concentrations at 1: 1 put into frozen pipe, the cryovial that branch installs is put on freezing, put into the cool chamber of programmed cooling instrument immediately; Cooling process is 0 ℃ of beginning, be down to-5 ℃ with 3 ℃/minute speed from 0 ℃, be down to-15 ℃ with 5 ℃/minute speed from-5 ℃, be down to-25 ℃ with 10 ℃ of/minute speed from-15 ℃, be down to-80 ℃, last balance 5 minutes with 20 ℃ of/minute speed from-25 ℃ again, after cooling is finished, directly sperm is taken out from the programmed cooling instrument, put into liquid nitrogen container immediately, in-198 ℃, preserve.
(4) seminal fluid that thaws: earlier cool chamber is mentioned on the liquid nitrogen liquid level, balance 3-5 minute, take out 1 frozen pipe with tweezers, be positioned over rapidly then in 42 ℃ of water-baths of preprepared and thaw;
(5) sea-water activated sperm use in the back that thaws, and places the microscopically observation that the sperm quality analytical system connects;
(6) sperm quality detects: utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer-aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.The detection analysis result shows: preservation effect is followed successively by Hank ' s+10%DMSO (sperm viability of thawing is 94.718%), TS-2+8%DMSO (sperm viability of thawing is 93.812%), TS-19+10%DMSO (sperm viability of thawing is 88.620%), Cortland+10%DMSO (sperm viability of thawing is 82.527%).
Embodiment 2
(1) select the rock salmon of body weight 4.5kg, the human chorionic gonadotrophin (HCG) of dorsal muscles injection 150IU/kg, during observe the variation of milter sperm volume;
(2) proportioning of antifreeze: antifreeze be Hank ' s, TS-2 and Cortland4 kind base fluid respectively with weight content 6,8,10,12,14 and 16%DMSO carry out proportioning, it is stand-by that the liquid that proportioning is good is positioned over 4 ℃ of refrigerators, presses 1: 1.5 part by weight with seminal fluid and antifreeze proportioning;
(3) the programmed cooling instrument is lowered the temperature: open computer and liquid nitrogen pump, open the programmed cooling instrument, make cooling storehouse temperature in 15～20 minutes, be down to 0 ℃, wait for application of sample, 4 kinds of base fluids are mixed with sperm with the DMSO mixed liquor of variable concentrations at 1: 1 put into frozen pipe, the cryovial that branch installs is put on freezing, put into the cool chamber of programmed cooling instrument immediately; Cooling process is 0 ℃ of beginning, be down to-5 ℃ with 3 ℃/minute speed from 0 ℃, be down to-15 ℃ with 5 ℃/minute speed from-5 ℃, be down to-25 ℃ with 10 ℃ of/minute speed from-15 ℃, be down to-80 ℃, last balance 5 minutes with 20 ℃ of/minute speed from-25 ℃ again, after cooling is finished, directly sperm is taken out from the programmed cooling instrument, put into liquid nitrogen container immediately, in-198 ℃, preserve.
(4) seminal fluid that thaws: earlier cool chamber is mentioned on the liquid nitrogen liquid level, balance 3-5 minute, take out 1 frozen pipe with tweezers, be positioned over rapidly then in 42 ℃ of water-baths of preprepared and thaw;
(5) sea-water activated sperm use in the back that thaws, and places the microscopically observation that the sperm quality analytical system connects;
(6) sperm quality detects: utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer-aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.The detection analysis result shows: preservation effect is followed successively by Hank ' s+10%DMSO (sperm viability of thawing is 94.718%), TS-2+8%DMSO (sperm viability of thawing is 93.812%), TS-19+10%DMSO (sperm viability of thawing is 88.620%), Cortland+10%DMSO (sperm viability of thawing is 82.527%).
Embodiment 3
(1) select the rock salmon of body weight 4.5kg, the human chorionic gonadotrophin (HCG) of dorsal muscles injection 150IU/kg, during observe the variation of milter sperm volume;
(2) proportioning of antifreeze: antifreeze be Hank ' s and Cortland4 kind base fluid respectively with weight content 6,8,10,12,14 and 16%DMSO carry out proportioning, it is stand-by that the liquid that proportioning is good is positioned over 4 ℃ of refrigerators, presses 1: 1.5 part by weight with seminal fluid and antifreeze proportioning;
(3) the programmed cooling instrument is lowered the temperature: open computer and liquid nitrogen pump, open the programmed cooling instrument, make cooling storehouse temperature in 15～20 minutes, be down to 0 ℃, wait for application of sample, 4 kinds of base fluids are mixed with sperm with the DMSO mixed liquor of variable concentrations at 1: 1 put into frozen pipe, the cryovial that branch installs is put on freezing, put into the cool chamber of programmed cooling instrument immediately; Cooling process is 0 ℃ of beginning, be down to-5 ℃ with 3 ℃/minute speed from 0 ℃, be down to-15 ℃ with 5 ℃/minute speed from-5 ℃, be down to-25 ℃ with 10 ℃ of/minute speed from-15 ℃, be down to-80 ℃, last balance 5 minutes with 20 ℃ of/minute speed from-25 ℃ again, after cooling is finished, directly sperm is taken out from the programmed cooling instrument, put into liquid nitrogen container immediately, in-198 ℃, preserve.
(4) seminal fluid that thaws: earlier cool chamber is mentioned on the liquid nitrogen liquid level, balance 3-5 minute, take out 1 frozen pipe with tweezers, be positioned over rapidly then in 42 ℃ of water-baths of preprepared and thaw;
(5) sea-water activated sperm use in the back that thaws, and places the microscopically observation that the sperm quality analytical system connects;
(6) sperm quality detects: utilize the existing sperm quality analytical system in laboratory (CASA) to carry out the computer-aided detection of sperm quality, utilize modern Computer Recognition Technology and image processing techniques, the sound attitude feature of sperm is carried out comprehensive quantitative analysis.The detection analysis result shows: preservation effect is followed successively by Hank ' s+10%DMSO (sperm viability of thawing is 94.718%), TS-2+8%DMSO (sperm viability of thawing is 93.812%), TS-19+10%DMSO (sperm viability of thawing is 88.620%), Cortland+10%DMSO (sperm viability of thawing is 82.527%).
The above embodiment is one of the present invention's preferred embodiments of being applied to the rock salmon fish, and its method also can be extended to the freezing and storing method of other fish sperm.
Claims (2)
1.
A kind of rock salmon sperm freezing is preserved and Activiation method, comprise the collection of rock salmon sperm, dilution, cooling and freezing, it is characterized in that: described freezing preservation is that the collection of rock salmon sperm is diluted and balance sperm with dilution and antifreeze later on, adopt again segmentation progressively falling temperature method carry out freezing preservation; Described Activiation method is with the sea-water activated sperm of the seminal fluid that thaws, and carries out sperm quality at last and detects;
With dilution and antifreeze sperm being diluted with balance is that rock salmon seminal fluid and dilution and the antifreeze proportioning part by weight by 1:1～2 is diluted; Described dilution is Hanks ', TS-2; Described antifreeze is that weight content is 8%～10% methyl-sulfoxide (DMSO);
Described segmentation progressively falling temperature method to carry out freezing preservation be to utilize the container that liquid nitrogen is housed, control rate of temperature fall by the control sample apart from the height of liquid nitrogen surface, temperature is by 0 ℃ of beginning, be down to-5 ℃ with 3 ℃/minute speed from 0 ℃, be down to-15 ℃ with 5 ℃/minute speed from-5 ℃, be down to-25 ℃ with 10 ℃ of/minute speed from-15 ℃, be down to-80 ℃ with 20 ℃ of/minute speed from-25 ℃ again, last balance 3～8 minutes, after cooling is finished, sperm is taken out from the programmed cooling instrument, put into liquid nitrogen container, in-198 ℃, preserve.
2.
Rock salmon sperm freezing according to claim 1 is preserved and Activiation method, it is characterized in that: it is to utilize Computer Recognition Technology and image processing techniques that described sperm quality detects, dynamic and static nature to sperm carries out comprehensive quantitative analysis, detects movement locus, movement rate, distribution, sperm profile, sperm quantity, viscosity and the vigor of sperm.
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| CN102870763A (en) * | 2011-07-11 | 2013-01-16 | 广东海洋大学 | Cryopreservation method of Lutjanus erythropterus sperms |
| CN110786321A (en) * | 2019-12-05 | 2020-02-14 | 广西壮族自治区水产科学研究院 | Freeze preservation method for pseudosciaena crocea sperms |
| CN114258911B (en) * | 2022-03-01 | 2022-06-14 | 中山大学 | Cryopreservation solution and cryopreservation method for sparus latus sperms |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
| CN1951183A (en) * | 2006-11-28 | 2007-04-25 | 厦门大学 | Daiquzu pseudosciaena crocea and minyuedongzu cultured pseudosciaena crocea cross breeding method |
| CN101703039A (en) * | 2009-11-10 | 2010-05-12 | 淮海工学院 | Sperm cryopreservation method of Charybdisjaponica |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1596670A (en) * | 2004-07-27 | 2005-03-23 | 中国水产科学研究院黄海水产研究所 | Practicalization method for frozen preserving sperm of fish |
| CN1951183A (en) * | 2006-11-28 | 2007-04-25 | 厦门大学 | Daiquzu pseudosciaena crocea and minyuedongzu cultured pseudosciaena crocea cross breeding method |
| CN101703039A (en) * | 2009-11-10 | 2010-05-12 | 淮海工学院 | Sperm cryopreservation method of Charybdisjaponica |
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