CN102726369A - Ultralow temperature cryopreservation application method of Epinephelus oanceolutus sperms - Google Patents
Ultralow temperature cryopreservation application method of Epinephelus oanceolutus sperms Download PDFInfo
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Abstract
An ultralow temperature cryopreservation application method of Epinephelus oanceolutus sperms belongs to the technical fields of the fish cryobiology and fish sperm ultralow temperature cryopreservation. The method comprises the steps of semen acquisition, sperm vitality detection, cryopreservation solution (ELS) preparation, semen dilution, sperm ultralow temperature cryopreservation, sperm unfreezing and detection, and frozen sperm fertilization. According to the method, the average frozen sperm vitality reaches 83.67%, there are no substantial differences between the frozen sperms and fresh sperms, and the frozen sperms can be hybridized with Epinephelus fuscogutatus. The method utilizes a diluent which has the advantages of simple formula and low cost and allows the Epinephelus oanceolutus semen to be massively cryopreserved, so problems of non-synchronous male and female maturation in artificial breeding of the Epinephelus oanceolutus, large geographical isolation in the hybridization with other groupers, and insufficient semen are solved.
Description
Technical field
The invention belongs to fish cryobiology and fish sperm superfreeze and preserve technical field, be specifically related to a kind of dragon wholesale sperm super-low temperature freezing store method.
Background technology
Dragon wholesale (Epinephelus lanceolatus) belongs to Perciformes (Perciformes), ester section (Serranidae), Epinephelus (Epinephalus), is water warm, middle lower floor coral fishes; Fast growth, 1 age fish can grow into 1.5-3.0kg, grow into 5-6kg 2 ages; Maximum can grow to about 2.7m; 440kg is build the maximum in the ablen, so also be called as " spot king ".Be distributed in Micronesia, middle part, eastern bank to the Pacific Ocean, Africa, the Indian Ocean, reach Australia in the south, marine site, the South Sea (Nansha Islands) is originated in China, but quantity is rare.The dragon wholesale is propagated artificially and is started from last century end, and artificial propagation and seed rearing technology are still immature, propagate the seed lazy weight artificially, have limited the development of epinephelus lanceolatus fish culture.
Because the dragon wholesale has the fast advantage of growth, for the good genetic resources of exploitation grouper, cultivate the fast new varieties of culturing of growth, improve culture benefit and have great importance.But dragon wholesale has hermaphroditism, and protogynous characteristics have only the oldestly in the same colony, and individual maximum fish talent and character is converted into male.This characteristic causes certain difficulty for artificial propagation, production and the crossbreeding of dragon wholesale, and therefore research dragon wholesale sperm super-low temperature freezing is preserved technology, and can effectively solve is a problem.Only aspect the influence of dragon wholesale sperm viability, (Daya Gulf aquatic products experimental center, Guangdong Province, Liang Weifeng etc., 2009) have been carried out studying both at home and abroad at present in environmental factor; But dragon wholesale sperm super-low temperature freezing is preserved, and utilized frozen sperm to carry out the research of hybrid breeding and breeding aspect, also be not reported.
Summary of the invention
The technical problem that the present invention will solve provides a kind of dragon wholesale sperm super-low temperature freezing store method; To solve in the southern and northern grouper hybridization; Male and female fish maturation is asynchronous, the breed geographic isolation is bigger; The south fish are cultured problems such as difficulty in the north, and dragon wholesale sperm freezing to preserve survival rate low, problem such as be difficult to widely apply aborning.
Technical scheme:
The present invention realizes through following technical scheme:
1, the collection of seminal fluid: at the integrated ripe seminal fluid of manual pick mating season; Utilize the wholesale of 50ppm eugenol anesthesia dragon; The manual compression belly flows out the milky seminal fluid, utilizes syringe to draw seminal fluid, prevents that urine and seawater from sneaking into; The seminal fluid of gathering is injected the 100ml vial, the vial of collecting seminal fluid is placed in the incubation chamber of ice cube;
2, sperm viability detects: the libation at an ancient wedding ceremony is got a little sperm and is coated on the slide, drip one sea-water activated, examine under a microscope sperm viability, vigor reaches more than 90% can carry out freezing preservation;
3, freezing preservation liquid (ELS) preparation: in distilled water, add 60g/L glucose (Glucose), 10g/L NaCl, 0.5g/L NaHCO
3, to add volume ratio again be 10%DMSO and 10% hyclone (FBS);
4, sperm dilution:, be placed on the incubation chamber balance 5min of ice cube with freezing preservation liquid and seminal fluid mixed with 1: 1;
5, sperm super-low temperature freezing is preserved: utilize the 1ml liquid-transfering gun will dilute the cryovial that the back seminal fluid injects 2ml; Every pipe adds dilution seminal fluid 1ml; Again cryovial is suspended at-the liquid nitrogen steam of 60--80 ℃ in balance 10min, sink to freezing preservation in the liquid nitrogen (196 ℃) then;
6, sperm thaws and detects: cryovial is taken out from liquid nitrogen; Balance 1min in the liquid nitrogen steam puts into 37 ℃ of water-baths with cryovial and thaws, and gets a little back seminal fluid that thaws and spreads upon on the slide; Drip one sea-water activated, examine under a microscope sperm viability;
7, frozen sperm fertilization: the back sperm viability of will thawing reaches in the lithosporic fish-egg that 80% above sperm joins collection, and smart ovum ratio is 1: 200ml, and utilize glass bar to mix gently, leave standstill 5min, add 2 times of seawater and be fertilized to the ovum amount.
The present invention's beneficial effect compared with prior art:
1, invents a kind of new sperm freezing and preserved formula of liquid (ELS:60g/L glucose+10g/L NaCl+0.5g/L NaHCO
3+ 10%DMSO+10% hyclone), have the advantages that simple, easy to operate, the freezing preservation sperm of composition is big, survival rate is high;
2, the sperm freezing survival rate is high, utilizes freezing the preservations dragon of the inventive method sperm survival rate of buying wholesale to reach 83.76%, does not have significant difference with frozen sperm survival rate not.
3, solved the problem that asynchronous, the various grouper geographic isolation of male and female fish maturation is far away in the grouper crossbreeding, milter quantity is few, sperm volume is not enough.
4, the seminal fluid reserve capacity is big, and each freezing reserve capacity can reach more than the 50ml, can be widely used in aspects such as grouper artificial propagation, crossbreeding.
The practical implementation method:
Preserve and the next further explanation of exomixis the present invention below in conjunction with dragon wholesale sperm freezing.
1, the collection of seminal fluid: utilize 6 of the dragon wholesale milters that new villages and small towns, Lingshui, Hainan Province county (Hainan blue ocean science culture company) cultivate, the long 1.3-1.6m of body, body weight 35-40kg.At the integrated ripe seminal fluid of mating season in April, 2012 manual pick.Utilize the anesthesia dragon wholesale of 50ppm eugenol, the manual compression belly flows out the milky seminal fluid, utilizes the 50ml syringe to draw seminal fluid, prevents that urine and seawater from sneaking into.The seminal fluid of gathering is injected the 100ml vial, the vial of collecting seminal fluid is placed in the incubation chamber of ice cube, altogether collecting semen 55ml.
2, sperm viability detects: libation at an ancient wedding ceremony extracting extract smear, drip sea-water activatedly, and in the microscopically detection, sperm viability reaches 93.33 ± 2.89%.
3, freezing preservation liquid (ELS) preparation: in distilled water, add 60g/L glucose (Glucose), 10g/L NaCl, 0.5g/L NaHCO
3, to add volume ratio again be 10%DMSO and 10% hyclone (FBS);
3, sperm freezing is preserved: utilize sperm freezing to preserve liquid (ELS) with 1: 1 dilution proportion seminal fluid; Utilize liquid-transfering gun to draw the cryovial that dilution seminal fluid 1ml adds 2ml; Cryovial is suspended at-the liquid nitrogen steam of 60--80 ℃ in balance 10min, sink to freezing preservation in the liquid nitrogen (196 ℃) then.Freezing preservation seminal fluid 110 is managed (110ml) altogether.
4, sperm thaws and detects: cryovial is taken out from liquid nitrogen, and balance 1min in the liquid nitrogen steam puts into 37 ℃ of water-baths with cryovial and thaws.Get a little thaw the back seminal fluid spread upon on the slide, drip one sea-water activated, examine under a microscope sperm viability.The back sperm viability of thawing reaches 83.76 ± 7.45%.
5, frozen sperm fertilization and hybrid breeding: blue ocean science is cultured company's freezing preservation dragon wholesale seminal fluid 110 pipes (110ml) in Hainan, and frozen semen is transported to Shandong Mingbo Aquatic Product Co., Ltd., Laizhou, gathers the garrupa mature egg 800ml that the said firm cultivates; Dragon wholesale sperm 4 pipes (4ml) thaw; Add in the ovum of gathering, stir mixing gently, adopt the dry method fertilization with glass bar; After leaving standstill 5ml, add 2 times to the seawater of ovum.Place little net to wash fertilized egg afterwards, flush away ovum liquid is put into 22 ℃ of seawater with fertilized egg and is hatched.Fertilization rate reached to 94.56%, incubation rate reaches 62.5%, obtains eyed eggs 500ml, hatches about fry 400,000 tails.
Claims (7)
1. a dragon wholesale sperm super-low temperature freezing is preserved application method; Comprise: sperm collection, sperm viability detection, the preparation of freezing preservation liquid, sperm dilution, sperm super-low temperature freezing are preserved, sperm thaws and detection, frozen sperm are fertilized, and it is characterized in that the preparation of sperm freezing preservation liquid: in distilled water, add 60g/L glucose, 10g/LNaCl, 0.5g/L NaHCO
3, 10% hyclone and 10% dimethyl sulfoxide (DMSO) be formulated.
2. dragon wholesale sperm super-low temperature freezing according to claim 1 is preserved application method, it is characterized in that sperm utilizes the wholesale of 50ppm eugenol anesthesia dragon when gathering, and manual compression belly collecting semen prevents that urine and seawater from sneaking into.
3. dragon wholesale sperm super-low temperature freezing according to claim 1 is preserved application method, and dragon wholesale sperm viability can be used for freezing preservation at the seminal fluid more than 90% when it is characterized in that the sperm viability detection.
4. dragon wholesale sperm super-low temperature freezing according to claim 1 is preserved application method, it is characterized in that described sperm dilution is that freezing preservation liquid mixes with 1: 1 dilution proportion with seminal fluid, is placed on the incubation chamber balance 5min of ice cube.
5. dragon wholesale sperm super-low temperature freezing according to claim 1 is preserved application method; It is characterized in that described sperm super-low temperature freezing saves as the cryovial with dilution seminal fluid 1ml injection 2ml; Cryovial is placed-the liquid nitrogen steam balance 10min of 60--80 ℃, directly cryovial is dropped into freezing preservation in the liquid nitrogen.
6. preserve application method according to the said dragon wholesale sperm super-low temperature freezing of claim 1, it is characterized in that described sperm thaws and detects to the sperm freezing pipe is put into 37 ℃ of water-baths thaws frozen sperm vigor average out to 83.67% behind the microscopy.
7. preserve application method according to the said dragon wholesale of claim 1 sperm super-low temperature freezing; It is characterized in that described frozen sperm fertilization mixes for the sperm that will thaw adds in the unfertilized egg; Smart ovum ratio is 1: 200ml adds 2 times of seawater fertilizations to ovum after leaving standstill 5min.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102986652A (en) * | 2012-12-25 | 2013-03-27 | 中国水产科学研究院黑龙江水产研究所 | Anti-freezing liquid for preserving pink salmon sperm at ultralow temperature in freezing manner and method for preserving pink salmon sperm at ultralow temperature by utilizing same |
| CN104304234A (en) * | 2014-09-17 | 2015-01-28 | 中山大学 | Freezing protection solution and preservation method of Plectropomus leopardus sperms |
| CN104686503A (en) * | 2015-03-24 | 2015-06-10 | 海南大学 | Method for freezing garrupa sperms in batch |
| CN104957130A (en) * | 2015-07-15 | 2015-10-07 | 福建师范大学 | Method for storing goldfish eggs at low temperature |
| CN110326610A (en) * | 2019-07-19 | 2019-10-15 | 大连海洋大学 | Sea cucumber sperm cryopreservation method |
| 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 |
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| CN102077823A (en) * | 2010-11-16 | 2011-06-01 | 佛山市生生水产股份有限公司 | Method for cryopreservation of semen of siniperca scherzeri and artificial insemination |
| CN102273439A (en) * | 2011-09-06 | 2011-12-14 | 中国水产科学研究院黄海水产研究所 | Cryopreservation and application method of Convict grouper sperms |
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Patent Citations (3)
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| 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 |
| CN102077823A (en) * | 2010-11-16 | 2011-06-01 | 佛山市生生水产股份有限公司 | Method for cryopreservation of semen of siniperca scherzeri and artificial insemination |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102986652A (en) * | 2012-12-25 | 2013-03-27 | 中国水产科学研究院黑龙江水产研究所 | Anti-freezing liquid for preserving pink salmon sperm at ultralow temperature in freezing manner and method for preserving pink salmon sperm at ultralow temperature by utilizing same |
| CN104304234A (en) * | 2014-09-17 | 2015-01-28 | 中山大学 | Freezing protection solution and preservation method of Plectropomus leopardus sperms |
| CN104686503A (en) * | 2015-03-24 | 2015-06-10 | 海南大学 | Method for freezing garrupa sperms in batch |
| CN104686503B (en) * | 2015-03-24 | 2016-08-24 | 海南大学 | A kind of method of frozen in batches cabrilla sperm |
| CN104957130A (en) * | 2015-07-15 | 2015-10-07 | 福建师范大学 | Method for storing goldfish eggs at low temperature |
| CN110326610A (en) * | 2019-07-19 | 2019-10-15 | 大连海洋大学 | Sea cucumber sperm cryopreservation method |
| CN110326610B (en) * | 2019-07-19 | 2021-09-24 | 大连海洋大学 | Ultralow temperature cryopreservation method for sea cucumber sperms |
| 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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