CN103348932B - 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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- CN103348932B CN103348932B CN201310212970.3A CN201310212970A CN103348932B CN 103348932 B CN103348932 B CN 103348932B CN 201310212970 A CN201310212970 A CN 201310212970A CN 103348932 B CN103348932 B CN 103348932B
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- paralichthys
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- 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
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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 field of marine biotechnology, specifically the test-tube method of interspecific cross of the summer flounder sperm of a kind of lefteye flounder ovum and freezen protective.
Background technology
Crossbreeding implements the also easy a kind of breeding method taken effect than being easier to, and it utilizes existing kind or species to hybridize, and selects the assortment of genes meeting people's needs from its offspring.Select the species of geographical distant race as hybrid strain, the polymorphism of hybrid generation can be increased, being easy to occur varied type from their filial generation, therefrom obtaining satisfactory newtype than being easier to.Lefteye flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus) are important mariculture fingerlings, and be distributed in the Pacific Ocean and the Atlantic coast respectively, although belong to same genus, geographical distribution is distant.Lefteye flounder (♀) and summer flounder (♂), as parent, by artificial insemination method, obtain growth, grow normal hybrid generation.The characteristics such as it is fine and smooth delicious, nutritious that hybrid generation inherits its parent's meat, and edibility is high, show that growth is fast, Acclimation temperature scope is wide propagating artificially in process simultaneously, particularly the advantage such as high temperature resistant.But lefteye flounder and summer flounder have different biological propertyes often in artificial culture process gonad development asynchronous, simultaneously lefteye flounder also often cultivates from summer flounder the difficult problem that this all causes interspecific cross sperm, ovum is difficult to synchronization gain in different regions.
Summary of the invention
The object of the invention is to provide one summer flounder to freeze essence and the test-tube method of lefteye flounder interspecific cross.
The technical solution used in the present invention is for achieving the above object:
One summer flounder freezes essence and the test-tube method of lefteye flounder interspecific cross, the summer flounder sperm of long-term Ultra-cryofreezing preservation is thawed through two step water-baths, dissolve after the summer flounder freeze in essence the dilution adding its 3 times of volumes, centrifugal collecting precipitation, then carry out artificial insemination with lefteye flounder ovum; Wherein, dilution is NaCl, KH2PO4 and water composition.
Further, the ratio that itself and anti frozen liquid are 1:3 is by volume shaken gently by the summer flounder sperm gathering essence vigor more than 85% breeding period in trash ice, abundant mixing balances 5-6min; Then be cooled to-150 DEG C with the rate of temperature fall of-10 DEG C/min, be placed in liquid nitrogen and carry out long-term Ultra-cryofreezing preservation, obtain the summer flounder sperm of long-term Ultra-cryofreezing preservation.
Described anti frozen liquid is made up of antifreeze, antifreeze and dilution; The sucrose of wherein antifreeze is propane diols (PG), the antifreeze of 13% to be concentration be 30g/L.
Described two step water-bath solutions are first placed in 37 DEG C of water-baths shake 90s gently by freezing essence, is then placed in 18 DEG C of water-baths and shakes 30s gently, carry out two step water-bath solutions, melt in the ice chest being placed on 0-4 DEG C and preserve.
By described after two step water-baths are thawed the summer flounder freeze essence and mix with the ratio of dilution 1:3 by volume, with the centrifugal force 5min of 800g after mixing, then remove supernatant, obtain with dilute front same volume freeze sperm-suspension, be positioned over 0-4 DEG C of ice chest, stand-by.
By after water-soluble for described two steps thawing the summer flounder freeze essence and activate for the ratio of 1:40 mix by volume with nature seawater, inject lefteye flounder ovum after activating, mix gently, lefteye flounder ovum and summer flounder are frozen and smartly fully to contact, standing 5-10min; Then wash ovum, use bolting silk net filtration, the fertilized egg of collection is positioned in fresh seawater and rinses 3 ~ 5 times, leave standstill after rinsing, get floating ovum and hatch.
Institute of the present invention tool has the following advantages:
1. the present invention gathers sexual maturity summer flounder milter high-quality sperm through Ultra-cryofreezing preservation, then obtains the lefteye flounder ovum synchronous with it and carries out interspecific cross and then realize artificial insemination.Wherein, the Ultra-cryofreezing preservation technology of summer flounder sperm can be preserved summer flounder for a long time and frozen essence, application of can thawing at any time, everywhere.
2. during Ultra-cryofreezing preservation summer flounder sperm of the present invention with 13% propane diols (PG) for permeability antifreeze, sucrose 30g/L as taking permeability antifreeze, increase to freeze essence protected effect and effectively reduce toxicity.
3. adopt 37 DEG C and 18 DEG C of two step water-bath to thaw in Ultra-cryofreezing preservation summer flounder sperm course of defrosting of the present invention, avoid the too high damage to freezing essence and causing of cryopreservation tube environment temperature.
4. the present invention is frozen essence and is adopted 3 times of volume dilution liquid dilutions before for artificial insemination, the centrifugal force wash-out of 800g, and the toxicity reducing antifreeze does not destroy the structure of sperm simultaneously on the impact of freezing extract Iuality.
5. first adopt in artificial insemination procedures of the present invention a certain amount of seawater will freeze essence activate then pour in ovum, seawater activation freeze essence while again can further wash-out antifreeze on the impact of sperm viability;
6. the test-tube repeated good stability of the present invention, freeze well essence thaw after anabiosis rate high, activate after rate of motion 60-70%, fertilization rate at 50-70%, incubation rate 60-70%.
Embodiment
Embodiment 1
Gather the sperm containing the phase on the occasion of summer flounder reproduction, namely gather summer flounder sperm in parent fish rearing workshop, ocean, east, Yantai, and be stored in clean centrifuge tube, microscopic examination vigor, higher than 90%, is placed in and trash ice takes back laboratory carries out freezen protective;
The fresh spermatozoa of above-mentioned collection is shaken gently through being blended in (0 DEG C) in trash ice by volume ratio and the anti frozen liquid of 1:3, fully being mixed 3min, and is dispensed in 2ml cryopreservation tube, be then placed in cooling instrument 0 DEG C and balance 5min; Be cooled to-150 DEG C with the rate of temperature fall of-10 DEG C/min again, be dispensed into freezing storing box, be placed in liquid nitrogen container, carry out long-term Ultra-cryofreezing preservation, preserve and freeze essence 65 pipe;
Wherein, anti frozen liquid is made up of antifreeze, antifreeze and dilution; Dilution is mass concentration 8g/L NaCl, 1.5g/L KH
2pO
4and water; Antifreeze is the propane diols (PG) of 13%, and the sucrose of antifreeze to be concentration be 30g/L, namely anti frozen liquid is NaCl8g, KH in every premium on currency
2pO
41.5g, sucrose 30g, 13mL PG(V/V).
First configure dilution with distilled water, weigh NaCl8g and KH
2pO
4it is for subsequent use that then 1.5g is settled to 1L; Liquid configuration anti frozen liquid based on the dilution that employing has configured, wherein containing 13%DMSO(V:V) (namely 100mL anti frozen liquid is containing 13mL PG); Then by liquid based on the anti frozen liquid that configured, add sucrose 3.42g, be settled to 100mL.
The summer flounder of above-mentioned freezen protective is frozen essence employing 37 DEG C of water-baths of thawing to thaw, cryopreservation tube box is taken out from the layer frame of liquid nitrogen container, be positioned over and have in the incubation chamber of liquid nitrogen, from freezing storing box, take out cryopreservation tube fast with tweezers be placed in 37 DEG C of water-baths and shake about 90s gently, then cryopreservation tube is placed in 18 DEG C of water-baths and shakes about 30s gently, the ice chest being then placed in 0-4 DEG C is deposited;
After thawing freeze smart artificial insemination before be mixed in 10ml centrifuge tube with the volume ratio of 1:3 and dilution again, wash-out is carried out with the centrifugal force 5min of 800g, then supernatant is removed, what obtain same volume front with dilution freezes sperm-suspension, is positioned in 0-4 DEG C of ice chest and prepares at any time to carry out artificial insemination;
Add 100ml nature seawater in beaker, then add 2.5ml and freeze essence, mixing, pours into rapidly in 1kg high-quality lefteye flounder ovum, mixes gently, leaves standstill 5min; Then wash ovum, use bolting silk net filtration, the fertilized egg of collection be positioned in fresh seawater and rinse 3 ~ 5 times, fertilized egg is put into 2000ml graduated cylinder and leave standstill, get floating ovum and hatch, incubation rate 60-70%, seed () survival rate higher than 80% at abnormal the volt end.
Embodiment 2
In May, 2010, in March, 2011, in December, 2012, gather summer flounder sperm in parent fish rearing workshop, ocean, east, Yantai, microscopic examination vigor is higher than 90%.Fresh essence is placed in and trash ice takes back laboratory carries out freezen protective or the fresh spermatozoa of above-mentioned collection is directly carried out freezen protective at the scene.
Fresh essence is shaken gently through being blended in (0 DEG C) in trash ice by volume ratio and the anti frozen liquid of 1:3, fully being mixed 3min, and is dispensed in 2ml cryopreservation tube, is then placed in cooling instrument 0 DEG C and balances 5min; Be cooled to-150 DEG C with the rate of temperature fall of-10 DEG C/min again, be dispensed into freezing storing box, be placed in liquid nitrogen container, carry out long-term Ultra-cryofreezing preservation, preserve and freeze essence 65 pipe;
Wherein, anti frozen liquid is made up of antifreeze, antifreeze and dilution; Dilution is mass concentration 8g/L NaCl, 1.5g/L KH
2pO
4and water; Antifreeze is the propane diols (PG) of 13%, and the sucrose of antifreeze to be concentration be 30g/L, namely anti frozen liquid is containing NaCl8g, KH in every premium on currency
2pO
41.5g, sucrose 30g, 13mL PG(V/V).
The different year be kept in a liquid nitrogen container essence of freezing of preserving is transported to Yantai Tian Yuan aquatic products company, the summer flounder of above-mentioned freezen protective is frozen essence employing 37 DEG C of water-baths of thawing to thaw, cryopreservation tube box is taken out from the layer frame of liquid nitrogen container, be positioned over and have in the incubation chamber of liquid nitrogen, from freezing storing box, take out cryopreservation tube fast with tweezers be placed in 37 DEG C of water-baths and shake about 90s gently, then cryopreservation tube is placed in 18 DEG C of water-baths and shakes about 30s gently, the ice chest being then placed in 0-4 DEG C is deposited;
After thawing freeze smart artificial insemination before be mixed in 10ml centrifuge tube with the volume ratio of 1:3 and dilution again, wash-out is carried out with the centrifugal force 5min of 800g, then supernatant is removed, what obtain same volume front with dilution freezes sperm-suspension, is positioned in 0-4 DEG C of ice chest and prepares at any time to carry out artificial insemination;
Gather and obtain high-quality lefteye flounder ovum, by 100ml beaker in add about 1ml seawater, then add 5 μ L and freeze essence and add 1-2mL ovum simultaneously, mix gently; Mix gently, leave standstill 5min; Then wash ovum, use bolting silk net filtration, the fertilized egg of collection be positioned in fresh seawater and rinse 3 ~ 5 times, fertilized egg is put into 2000ml graduated cylinder and leave standstill, get floating ovum and hatch, the essence of freezing in each time does three Duplicate Samples simultaneously.Experimental result display 3 years of preserving continuously freeze polyspermism rate 60-90%, incubation rate 80-90%.
Claims (4)
1. one kind the summer flounder freeze essence with the test-tube method of lefteye flounder interspecific cross, it is characterized in that: the summer flounder sperm of long-term Ultra-cryofreezing preservation is thawed through two step water-baths, melt rear summer flounder and freeze in essence the dilution adding its 3 times of volumes, centrifugal collecting precipitation, then carry out artificial insemination with lefteye flounder ovum; Wherein, dilution is NaCl, KH
2pO
4form with water;
It is first be placed in 37 DEG C of water-baths shake 90s gently by freezing essence that described two step water-baths are thawed, and is then placed in 18 DEG C of water-baths and shakes 30s gently, carry out two step water-baths and thaw, melt in the ice chest being placed on 0-4 DEG C and preserve;
The summer flounder ratio of freezing essence and dilution 1:3 by volume after thawing through two step water-baths mix, with the centrifugal force 5min of 800g after mixing, then removes supernatant, obtain with dilute front same volume freeze sperm-suspension, be positioned over 0-4 DEG C of ice chest, stand-by.
2. freeze essence and the test-tube method of lefteye flounder interspecific cross by summer according to claim 1 flounder, it is characterized in that: the summer flounder sperm gathering essence vigor more than 85% breeding period by its with anti frozen liquid by volume for the ratio of 1:3 shake gently in trash ice, abundant mixing balance 5-6min; Then be cooled to-150 DEG C with the rate of temperature fall of-10 DEG C/min, be placed in liquid nitrogen and carry out long-term Ultra-cryofreezing preservation, obtain the summer flounder sperm of long-term Ultra-cryofreezing preservation.
3. freeze essence and the test-tube method of lefteye flounder interspecific cross by summer according to claim 2 flounder, it is characterized in that: described anti frozen liquid is made up of antifreeze 1, antifreeze 2 and dilution; Wherein antifreeze 1 be 13% propane diols (PG), antifreeze 2 for concentration be the sucrose of 30g/L.
4. freeze essence and the test-tube method of lefteye flounder interspecific cross by summer according to claim 1 flounder, it is characterized in that: the summer flounder ratio of freezing essence and dilution 1:3 by volume after thawing through two step water-baths is mixed, with the centrifugal force 5min of 800g after mixing, then supernatant is removed, what obtain same volume front with dilution freezes sperm-suspension, be positioned over 0-4 DEG C of ice chest, stand-by, then activate for the ratio of 1:40 mixes by volume with nature seawater, lefteye flounder ovum is injected after activating, mix gently, make lefteye flounder ovum and summer flounder freeze essence fully to contact, leave standstill 5-10min, then wash ovum, use bolting silk net filtration, the fertilized egg of collection is positioned in fresh seawater and rinses 3 ~ 5 times, leave standstill after rinsing, get floating 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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| 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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| 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN101884322A (en) * | 2010-07-20 | 2010-11-17 | 中国水产科学研究院黄海水产研究所 | Verasper moseri sperm cryopreservation method |
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Patent Citations (2)
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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 |
| CN101884322A (en) * | 2010-07-20 | 2010-11-17 | 中国水产科学研究院黄海水产研究所 | Verasper moseri sperm cryopreservation method |
Non-Patent Citations (3)
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| "三种重要海水鱼类胚胎程序化冷冻保存技术研究";于过才;《中国优秀硕士学位论文全文数据库》;20050315(第1期);第52-82页 * |
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| "海水鱼类精液超低温保存技术及其质量检测规范http://www.dxy.cn/bbs/topic/7606811?onlyHost=1";wangmengqiang;《丁香园》;20061125;第1-12页 * |
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