CN103348932A - Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus - Google Patents
Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus Download PDFInfo
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- CN103348932A CN103348932A CN2013102129703A CN201310212970A CN103348932A CN 103348932 A CN103348932 A CN 103348932A CN 2013102129703 A CN2013102129703 A CN 2013102129703A CN 201310212970 A CN201310212970 A CN 201310212970A CN 103348932 A CN103348932 A CN 103348932A
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- flounder
- paralichthys
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- dentatus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention belongs to the technical field of marine organisms, particularly a method for carrying out interspecies cross artificial insemination on an egg of paralichthys olivaceus and a semen of paralichthys dentatus, which is frozen for preservation. The semen of paralichthys dentatus, which is frozen for preservation at an ultralow temperature for a long time, is dissolved in water by two steps; after the semen of paralichthys dentatus is dissolved, diluent of which the volume is three times of that of the frozen semen of paralichthys dentatus is added into the frozen semen of paralichthys dentatus; after the frozen semen of paralichthys dentatus and the diluent are mixed, centrifugation is carried out for 5min; then supernate is removed to obtain frozen semen suspension with the same volume with the frozen semen of paralichthys dentatus before the frozen semen of paralichthys dentatus is diluted; the frozen semen and natural seawater are uniformly mixed according to the proportion of a volume ratio of 1:40 and the mixture is activated; after the mixture is activated, the egg of paralichthys olivaceus is injected and the mixture and the egg of paralichthys olivaceus are slightly and uniformly mixed to enable the egg of paralichthys olivaceus and the frozen semen of paralichthys dentatus to be sufficiently in contact; standing is carried out for 5 to 10min and egg washing is carried out to obtain a fertilized egg to carry out incubation. The invention establishes a cryopreservation technology for semen of paralichthys dentatus and a method for interspecies cross artificial insemination on the egg of paralichthys olivaceus, overcomes the difficulty of asynchrony of development of parental fishes of paralichthys dentatus and paralichthys olivaceus and provides a novel method and a novel idea for seawater fish cross breeding.
Description
Technical field
The invention belongs to the marine biotechnology field, the test-tube method of interspecific cross of the summer flounder sperm of specifically a kind of lefteye flounder ovum and freezing preservation.
Background technology
Crossbreeding is than a kind of breeding method that is easier to implement also to take effect easily, and it utilizes existing kind or species to hybridize, and selects the assortment of genes that meets people's needs from its offspring.Select the species of geographical distant race as hybrid strain, can increase the polymorphism of hybrid generation, be easy to varied type occur from their filial generation, therefrom obtain satisfactory newtype than being easier to.Lefteye flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus) are important mariculture fingerlings, are distributed in respectively the Pacific Ocean and the Atlantic coast, although belong to same genus, geographical distribution is distant.Lefteye flounder (♀) and summer flounder (♂) by artificial insemination method, obtain growth, the normal hybrid generation of growing as the parent.It is fine and smooth delicious, nutritious that hybrid generation has been inherited its parent's meat, and the characteristics such as edibility height show that growth is fast, the temperature wide accommodation simultaneously in propagating process artificially, particularly the advantage such as high temperature resistant.But lefteye flounder from the summer flounder have different biological propertyes often in the artificial culture process gonad development asynchronous, simultaneously lefteye flounder from the summer flounder often cultivate also in different regions that this all causes interspecific cross sperm, ovum to be difficult to the difficult problem of synchronization gain.
Summary of the invention
The object of the invention is to provide a kind of summer flounder to freeze smart and the test-tube method of lefteye flounder interspecific cross.
The technical solution used in the present invention is for achieving the above object:
A kind of summer, flounder froze smart and the test-tube method of lefteye flounder interspecific cross, the summer flounder sperm that long-term superfreeze is preserved thaws through two step water-baths, the rear summer of dissolving, flounder was frozen the dilution that adds its 3 times of volumes in the essence, and centrifugal collecting precipitation then carries out artificial insemination with the lefteye flounder ovum; Wherein, dilution is NaCl, and KH2PO4 and water form.
Further, gathering the summer flounder sperm of smart vigor breeding period more than 85% is ratio light shaking, the abundant mixing balance 5-6min in trash ice of 1:3 with itself and anti frozen liquid by volume; Then the rate of temperature fall with-10 ℃/min is cooled to-150 ℃, places liquid nitrogen to carry out long-term superfreeze and preserves, and obtains the summer flounder sperm that long-term superfreeze is preserved.
Described anti frozen liquid is comprised of antifreeze, antifreeze and dilution; Wherein antifreeze is that 13% propane diols (PG), antifreeze are that concentration is the sucrose of 30g/L.
Described two step water-bath solutions are to place first 37 ℃ of water-baths to shake gently 90s with freezing essence, then place 18 ℃ of water-baths to shake gently 30s, carry out two step water-bath solutions, melt in the ice chest that is placed on 0-4 ℃ and preserve.
With described after two step water-baths are thawed the summer flounder freeze smart and dilution by volume the ratio of 1:3 mixes, with the centrifugal 5min of the centrifugal force of 800g, then removes supernatant after mixing, obtain the sperm-suspension that freezes with the front equally volume of dilution, be positioned over 0-4 ℃ of ice chest, stand-by.
With described two steps water-soluble rear summer flounder of thawing freeze smart and nature seawater by volume for the ratio mixing of 1:40 activates, inject the lefteye flounder ovum after activating, mixing gently, make the lefteye flounder ovum with the summer flounder freeze essence and fully contact, leave standstill 5-10min; Then wash ovum, use the bolting silk net filtration, the fertilized egg of collecting is positioned over washes in the fresh seawater 3～5 times, leave standstill after the flushing, get the come-up ovum and hatch.
The present invention has following advantage:
1. the present invention gathers sexual maturity summer flounder milter high-quality sperm through the superfreeze preservation, then obtains the lefteye flounder ovum synchronous with it and carries out interspecific cross and then realize artificial insemination.Wherein, the superfreeze Techniques of preserving of summer flounder sperm can the long preservation summer flounder freeze essence, the application of can thawing at any time, everywhere.
Superfreeze of the present invention preserve the summer during flounder sperm take 13% propane diols (PG) as the permeability antifreeze, sucrose 30g/L is as take the permeability antifreeze, increase is to the protection effect of freezing essence and effectively reduce toxicity.
3. 37 ℃ and 18 ℃ two step water-bath of employing thawed in the superfreeze preservation summer flounder sperm course of defrosting of the present invention, avoided the too high damage that causes freezing essence of cryopreservation tube environment temperature.
4. the smart 3 times of volume dilution liquid dilutions of adopting are frozen in the present invention before being used for artificial insemination, the centrifugal wash-out of the centrifugal force of 800g, and the toxicity that reduces antifreeze is not destroyed the structure of sperm simultaneously on the impact of freezing extract Iuality.
5. at first adopt a certain amount of seawater will freeze smart the activation in the artificial Process of Insemination of the present invention and then pour in the ovum, seawater activation freeze smart in again further the wash-out antifreeze on the impact of sperm viability;
6. the repeated good stability of the artificial insemination of the present invention, it is high to freeze well the essence rear anabiosis rate that thaws, and rate of motion 60-70% after activating, fertilization rate be at 50-70%, incubation rate 60-70%.
Embodiment
Embodiment 1
Collection is contained the sperm of phase on the occasion of summer flounder reproduction, namely gathers summer flounder sperm in parent fish rearing workshop, ocean, east, Yantai, and is stored in the clean centrifuge tube, and the microscopic examination vigor is higher than 90%, places to take back the laboratory on the trash ice and carry out freezing preservation;
The fresh spermatozoa of above-mentioned collection through being blended in (0 ℃) light shaking in the trash ice, abundant mixing 3min by the volume ratio of 1:3 and anti frozen liquid, and being divided and installed in the 2ml cryopreservation tube, then place 0 ℃ of balance 5min of cooling instrument; Rate of temperature fall with-10 ℃/min is cooled to-150 ℃ again, divides to install to freezing storing box, places liquid nitrogen container, carries out long-term superfreeze and preserves, and preserves to freeze smart 65 pipes;
Wherein, anti frozen liquid is comprised of antifreeze, antifreeze and dilution; Dilution is mass concentration 8g/L NaCl, 1.5g/L KH
2PO
4And water; Antifreeze is 13% propane diols (PG), and antifreeze is that concentration is the sucrose of 30g/L, and namely anti frozen liquid is NaCl8g in every premium on currency, KH
2PO
41.5g, sucrose 30g, 13mL PG(V/V).
At first dispose dilution with distilled water, weigh NaCl8g and KH
2PO
41.5g it is for subsequent use then to be settled to 1L; Adopt the dilution that has disposed to dispose anti frozen liquid as basal liquid, wherein contain 13%DMSO(V:V) (being that the 100mL anti frozen liquid contains 13mL PG); The anti frozen liquid that then will configure adds sucrose 3.42g as basal liquid, is settled to 100mL.
The summer flounder of above-mentioned freezing preservation is frozen essence thaws and adopts 37 ℃ of water-baths to thaw, the cryopreservation tube box is taken out from the layer frame of liquid nitrogen container, be positioned in the incubation chamber that has liquid nitrogen, taking out fast cryopreservation tube with tweezers from freezing storing box places 37 ℃ of water-baths to shake gently about 90s, then cryopreservation tube is placed 18 ℃ of water-baths to shake gently about 30s, then place 0-4 ℃ ice chest to deposit;
Freeze before the smart artificial insemination again that volume ratio and the dilution with 1:3 is mixed in the 10ml centrifuge tube after thawing, centrifugal 5min carries out wash-out with the centrifugal force of 800g, then remove supernatant, obtain and the sperm-suspension that freezes that dilutes front same volume, be positioned over and prepare to carry out at any time artificial insemination in the 0-4 ℃ of ice chest;
Add the 100ml nature seawater in the beaker, then add 2.5ml and freeze essence, mixing is poured into rapidly in the 1kg high-quality lefteye flounder ovum, and mixing leaves standstill 5min gently; Then wash ovum, use the bolting silk net filtration, the fertilized egg of collecting is positioned over washes in the fresh seawater 3～5 times, fertilized egg is put into the 2000ml graduated cylinder leave standstill, get the come-up ovum and hatch, incubation rate 60-70%, seed (abnormal volt is at the end) survival rate is higher than 80%.
Embodiment 2
May in 2010 gathers summer flounder sperm in parent fish rearing workshop, ocean, east, Yantai March in 2011 in December, 2012, and the microscopic examination vigor is higher than 90%.Bright essence placed take back the laboratory on the trash ice and carry out freezing preservation or the fresh spermatozoa of above-mentioned collection is directly carried out freezing preservation at the scene.
Bright in smart being blended in (0 ℃) light shaking in the trash ice, abundant mixing 3min by the volume ratio of 1:3 and anti frozen liquid, and divide and install in the 2ml cryopreservation tube, then place 0 ℃ of balance 5min of cooling instrument; Rate of temperature fall with-10 ℃/min is cooled to-150 ℃ again, divides to install to freezing storing box, places liquid nitrogen container, carries out long-term superfreeze and preserves, and preserves to freeze smart 65 pipes;
Wherein, anti frozen liquid is comprised of antifreeze, antifreeze and dilution; Dilution is mass concentration 8g/L NaCl, 1.5g/L KH
2PO
4And water; Antifreeze is 13% propane diols (PG), and antifreeze is that concentration is the sucrose of 30g/L, and namely anti frozen liquid is to contain NaCl8g in every premium on currency, KH
2PO
41.5g, sucrose 30g, 13mL PG(V/V).
A different year essence of freezing of preserving that is kept in the liquid nitrogen container is transported to Yantai Tian Yuan aquatic products company, the summer flounder of above-mentioned freezing preservation is frozen essence thaws and adopts 37 ℃ of water-baths to thaw, the cryopreservation tube box is taken out from the layer frame of liquid nitrogen container, be positioned in the incubation chamber that has liquid nitrogen, taking out fast cryopreservation tube with tweezers from freezing storing box places 37 ℃ of water-baths to shake gently about 90s, then cryopreservation tube is placed 18 ℃ of water-baths to shake gently about 30s, then place 0-4 ℃ ice chest to deposit;
Freeze before the smart artificial insemination again that volume ratio and the dilution with 1:3 is mixed in the 10ml centrifuge tube after thawing, centrifugal 5min carries out wash-out with the centrifugal force of 800g, then remove supernatant, obtain and the sperm-suspension that freezes that dilutes front same volume, be positioned over and prepare to carry out at any time artificial insemination in the 0-4 ℃ of ice chest;
Gather to obtain high-quality lefteye flounder ovum, with 100ml beaker in add about 1ml seawater, then add 5 μ L and freeze essence and add simultaneously 1-2mL ovum, gently mixing; Mixing leaves standstill 5min gently; Then wash ovum, use the bolting silk net filtration, the fertilized egg of collecting is positioned over washes in the fresh seawater 3～5 times, fertilized egg is put into the 2000ml graduated cylinder leave standstill, get the come-up ovum and hatch, the essence of freezing in each time is done three Duplicate Samples simultaneously.Experimental result shows the polyspermism rate of the freezing 60-90% in 3 years that preserves continuously, incubation rate 80-90%.
Claims (6)
- One kind the summer flounder freeze smart and the test-tube method of lefteye flounder interspecific cross, it is characterized in that: the summer flounder sperm that long-term superfreeze is preserved thaws through two step water-baths, the rear summer of dissolving, flounder was frozen the dilution that adds its 3 times of volumes in the essence, and centrifugal collecting precipitation then carries out artificial insemination with the lefteye flounder ovum; Wherein, dilution is NaCl, KH 2PO 4Form with water.
- By summer claimed in claim 1 flounder freeze smart and the test-tube method of lefteye flounder interspecific cross, it is characterized in that: gathering the summer flounder sperm of smart vigor breeding period 85% or more is ratio light shaking, the abundant mixing balance 5-6min in trash ice of 1:3 by volume with itself and anti frozen liquid; Then the rate of temperature fall with-10 ℃/min is cooled to-150 ℃, places liquid nitrogen to carry out long-term superfreeze and preserves, and obtains the summer flounder sperm that long-term superfreeze is preserved.
- By summer claimed in claim 2 flounder freeze smart and the test-tube method of lefteye flounder interspecific cross, it is characterized in that: described anti frozen liquid is comprised of antifreeze, antifreeze and dilution; Wherein antifreeze is that 13% propane diols (PG), antifreeze are that concentration is the sucrose of 30g/L.
- By claim 1 or 2 described summer flounder freeze smart and the test-tube method of lefteye flounder interspecific cross, it is characterized in that: described two step water-bath solutions are to place first 37 ℃ of water-baths to shake gently 90s with freezing essence, then place 18 ℃ of water-baths to shake gently 30s, carry out two step water-bath solutions, melt in the ice chest that is placed on 0-4 ℃ and preserve.
- By summer claimed in claim 1 flounder freeze smart and the test-tube method of lefteye flounder interspecific cross, it is characterized in that: will be after two step water-baths be thawed the summer flounder freeze smart and dilution by volume the ratio of 1:3 mix, mix rear with the centrifugal 5min of the centrifugal force of 800g, then remove supernatant, obtain the sperm-suspension that freezes with the front same volume of dilution, be positioned over 0-4 ℃ of ice chest, stand-by.
- By claim 1 or 5 described summer flounder freeze smart and the test-tube method of lefteye flounder interspecific cross, it is characterized in that: with two steps the water-soluble rear summer flounder of thawing freeze and smartly activate for the ratio mixing of 1:40 by volume with nature seawater, inject the lefteye flounder ovum after activating, mixing gently, make the lefteye flounder ovum with the summer flounder freeze and smart fully contact, leave standstill 5-10min; Then wash ovum, use the bolting silk net filtration, the fertilized egg of collecting is positioned over washes in the fresh seawater 3～5 times, leave standstill after the flushing, get the come-up ovum and hatch.
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| CN201310212970.3A CN103348932B (en) | 2013-05-31 | 2013-05-31 | Method for carrying out interspecies cross artificial insemination on frozen semen of paralichthys dentatus and paralichthys olivaceus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103583439A (en) * | 2013-11-20 | 2014-02-19 | 文登市水产技术推广站 | Method for improving egg washing efficiency of bay scallops |
| CN104894061A (en) * | 2015-05-29 | 2015-09-09 | 中国科学院海洋研究所 | Backcross artificial insemination method for cryopreserved paralichthys dentatus sperms and paralichthys olivaceus ova |
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| CN101342192A (en) * | 2007-07-09 | 2009-01-14 | 中国水产科学研究院东海水产研究所 | Quick freezing conservation method for bastard halibut spermatozoon |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103583439A (en) * | 2013-11-20 | 2014-02-19 | 文登市水产技术推广站 | Method for improving egg washing efficiency of bay scallops |
| CN104894061A (en) * | 2015-05-29 | 2015-09-09 | 中国科学院海洋研究所 | Backcross artificial insemination method for cryopreserved paralichthys dentatus sperms and paralichthys olivaceus ova |
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