CN1221164C - Glass freezing preserving method for fish embryo - Google Patents

Abstract

The present invention relates to a vitrification freezing preserving method for fish embryos. In the method, toxic effect and vitrification performance of different agents are compared to screen out a vitrification liquid formula which is suitable for fish embryos in sea water; the period of embryonic development when vitrification is suitable to be processed is confirmed, a specific eluting solution is developed, and the vitrification freezing preserving technique for fish embryos is established by adopting the technical line of step-by-step balance, fast cooling and unfreezing. A vitrification freezing experiment is performed eight times on the embryos of bastard halibut in different periods of development, twenty freezing regeneration embryos are obtained, and the highest regeneration rate reaches 32 %. Fish fry are hatched by refreezing regeneration embryos is obtained five times in the experiments. Fourteen freezing embryos of twenty freezing regeneration embryos hatch fish fry, and the hatching rate reaches 70 %. The embryo propers of the fish fry hatched by freezing embryos are transparent and have the characteristics of normal shapes and no deformation. The technology of the present invention does not need expensive instruments or facilities, and has the advantages of simple operation, low cost and easy popularization. The present invention can be used for freezing preservation in ultralow temperature (196 DEG C) for fish embryos in sea water, such as bastard halibut, etc.

Description

The fish embryo vitrification freezing and storing method

Technical field:

The invention belongs to the animal embryo low temperature preservation technology in the cryobiology, is a kind of at the medium-term and long-term vitrification method of preserving fish embryo of liquid nitrogen (196 ℃).

Background technology:

Germ plasm resource is the important substance basis of aquaculture production, improved seeds cultivation and culture fishery sustainable development.China is the country of an aquatile germ plasm resource than horn of plenty, and rich and varied aquatile germ plasm resource and genetic diversity have played important effect for the fast development of China's culture fishery.Yet, because overfishing, unordered utilization and the artificial releasing etc. of fishery resources, caused the decline of some stock of fish and endangered, as the Changjiang river hilsa herring, trachidermus fasciatus etc.If the untimely safeguard measure of taking after the several years, will be difficult to find the genetic resources of many fish original seeds, breeding at occurring in nature.Therefore; set up the germplasm resource bank of important cultured fishes original seed, breeding and rare fish in imminent danger; germ plasm resource and the genetic diversity of these fish are got up with the form long preservation of living, become the important scientific and technological problem of demanding urgently capturing in fish germ plasma resource protection and the research of fish cryobiology.

Because fish-egg and embryo's volume (typically have a diameter from 1-7mm greatly, and mammality ovum diameter majority is in 200 μ m), water content is many, the egg membrane permeability is poor, the embryo by idiosome and two kinds of structures of yolk sac form, the yolk content height, so the superfreeze of fish embryo to preserve be a difficult problem of international fish low-temperature biological educational circles always.So far, do not set up the effective technology that the fish embryo superfreeze is preserved in the world as yet.Before the present invention made, the Harvey of University of Victoria: Victoria, Canada biology department (1982) was that material takes the lead in having carried out fish embryo low temperature preservation research with the zebra fish.Subsequently, domestic and international many scholars have carried out big quantity research to freshwater fish embryo cryopreservation preservation technology, and obtain some progress.On fish-egg and embryo cryopreservation method, the method for trying out has that segmentation is lowered the temperature at a slow speed, 3 kinds of segmentation fast cooling and vitrifying methods.Segmentation is lowered the temperature at a slow speed and is generally sample from (2-5 ℃/min) drop to the temperature of freezing point at a slow speed of room temperature, and then with (0.05-0.5 ℃/min) reduce to about-60 ℃ approximately of extremely slow speed, reduce to-85 ℃ with about 1-2 ℃/min again, stopped about 10 minutes, last fast cooling is-196 ℃ storage temperature extremely.(1989) such as Zhang X.S of low-temperature biological Engineering Room of Shanghai University of Science and Technology adopt this cool-down method at a slow speed when carrying out the preservation of carp embryo cryopreservation, from 16 pieces of embryos of 20 minutes of preservation liquid nitrogen, obtained the embryo of 3 pieces of resurrections, but regrettably, this experimental result but again repeats to come out in research subsequently.Second method is a segmentation fast cooling method.This method is exactly from 0 ℃ to-60 ℃ with the main difference of cooling at a slow speed, adopts the faster rate of temperature fall of 2-5 ℃/min.(1997) such as the Zhang Kejian of Shanghai Aquatic Products Univ. 9CN) adopt this cool-down method that grass carp and loach embryo have been carried out freezing preservation experiment, and obtain the successful example that indivedual embryos are brought back to life once in a while, but experimental result can not repeat.More than experiment is all because of forming ice crystal in the embryonic cell, and causes embryonic death.The third frozen cooling method is exactly a vitrifying freeze process.This method makes in the cell and crosses freezing process with extracellular liquid, and directly form vitreous solid by means of extremely fast cooling, thereby avoids forming ice crystal in the cell, reduces the damage that ice crystal forms pair cell, keeps cyto-architectural integrality.(1997) such as Chao N of Taiwan aquatic products research institute have compared the influence of several vetrifying solutions to the freezing preservation of zebrafish embryo, the effect that shows DAP2B (2M DMSO+1M acetamide+3M propane diols) is better than the effect of VSI (20.5%DMSO+15.5% acetamide+10% propane diols), but does not obtain the embryo of freezing resurrection.The Zhang of Britain Luton university and Rawson (1996) have studied the feasibility that the zebrafish embryo glass freezing is preserved, show that it is 3Mol/L that butanediol can form vitrified least concentration, the mixture of several different antifreezes can form vitrifying, the back embryo morphology is normal although thaw, and does not obtain the zebrafish embryo that brings back to life.(1998) primary studies such as Hagedorn in the U.S. Washington country zoo permeability barrier of zebrafish embryo to antifreeze, but do not obtain the fish embryo of freezing resurrection.In " Cryopreservation in Aquatic Species " this authoritative monograph that the TierschT and Mazik doctor PM (2000) at U.S. Louisiana state university agricultural center collaborates, classify the freezing preservation of fish embryo one of as difficult problem of not capturing as yet in the world so far.(2001) such as Zhang Longzhen of domestic the Changjiang river aquatic products research institute have observed the time-to-live of loach embryo in different glass liquid, several vitrified antifreeze solution that form have therefrom been filtered out, the embryo can reach 70min at the time-to-live in these vetrifying solutions, but does not obtain the fry of freezing resurrection.Therefore,, do not succeed as yet so far, do not set up practicable glass freezing preservation technology yet although some scholars have carried out many researchs to freshwater fish embryo's vitrifying technology.And relevant seawater fish embryo's freezing preservation, particularly lefteye flounder and perch embryo's glass freezing is preserved technology, does not all find any document and patent report.

Summary of the invention:

At fish embryo have volume big (diameter is 1-7mm), water content is many, the egg membrane permeability is poor, the embryo by idiosome and two kinds of structures of yolk sac form, characteristics such as yolk content height, the purpose of patent of the present invention is by setting up fish embryo glass freezing technology, solution forms ice crystal in the embryonic cell in the frozen cooling process, a difficult problem that causes embryonic death, thereby capture the technical barrier that the fish embryo superfreeze is preserved, for set up the fish embryo freezer, the long preservation fish germ plasma resource provides technological means.

The present invention realizes according to following operating technology: embryo collection, and the qualified back of microscopy is cultured to body segment and uses during the apparition in culture dish; With vitrifying base soln (BS2) dilution antifreeze 1,2-propane diols (being abbreviated as PG) and methyl alcohol (being abbreviated as MeOH) mixture, be mixed with the vetrifying solution (FVS1-FVS4) of variable concentrations, it consists of 50-67%BS2,20-30%PG and 13-20%MeOH; The fish embryo that will be in early stage in body segment apparition-membrane respectively soaked in the vetrifying solution that concentration increases progressively 9-11 minute step by step, froze forward horizontal stand; The embryo that balance is good changes the plastics straw over to, seals the back and directly carries out freezing preservation in the input liquid nitrogen; In the time of need thawing, carry out quick-thawing after straw taken out from liquid nitrogen.Embryo after will thawing was placed in the eluent wash-out 10 minutes, and eluent is the solution that adds 0.125Mol/L sucrose in BS2; Then, adding the sterilization seawater cultivates.

Determining of embryo collection and suitable developmental stage: gather fish embryo, handle the embryonic development period of determining suitable glass freezing by antifreeze, showing that embryo from the body segment apparition to the tail bud phase is all suitable carries out glass freezing and preserves.

The preparation of vitrifying base soln (BS2) (being the weight ratio in the 100ml water): NaCl 2.472%, KCl 0.086%, CaCl ₂.2H ₂O 0.146%, MgCl ₂.6H ₂O 0.486%, NaHCO ₃0.019%.

Antifreeze: be 1,2-propane diols (PG) and methyl alcohol (MeOH), its concentration is respectively 20-30% and 13-20%.

The preparation of vetrifying solution: the prescription of 4 kinds of vetrifying solutions that use among the present invention is respectively: FVS1 contains BS2 67%, PG 20% and MeOH 13%; FVS2 contains BS2 60%, PG 24% and MeOH 16%; FVS3 contains BS2 55%, PG 27% and MeOH 18%; FVS4 contains BS2 50%, PG 30% and MeOH20%.

Embryo's the forward horizontal stand that freezes is handled: the embryo is successively placed on contains 25%, 33%, carry out Balance Treatment, per step balance about 9-11 minute, 45-55 minute altogether in the equilibrium liquid of 50%, 67% and 100% FVS1 (or FVS2, FVS3, FVS4) vetrifying solution.The equilibrium liquid of above-mentioned 5 kinds of variable concentrations dilutes FVS1 (or FVS2, FVS3, FVS4) with the BS2 base soln and obtains.

Glass freezing: in the end grain is 3mm together with about 250ul vetrifying solution suction diameter surplus the embryo 10 that balance is good in the one-level vetrifying solution, length is the straw of 10cm, after hot-press sealed,, straw is dropped into (196 ℃) freezing preservation in the liquid nitrogen rapidly with the above rate of temperature fall of 2000 ℃/min.

The preparation of eluent: add sucrose in base soln BS2, concentration is 0.125Mol/L.

Thaw and cultivate: frozen straw is taken out from liquid nitrogen, put into 39-40 ℃ of water-bath rapidly, make its quick-thawing, straw is moved out to time of entering water-bath in 1-2 second from liquid nitrogen.When treating that embryo's milky is about to disappear straw is taken out from water-bath, cut off and seal, with the sucrose 2ml of 0.125M the embryo in the straw is flushed to and carries out wash-out in the culture dish, the time is 10min; Adding the sterilization seawater cultivates.

With the prior art contrast, the technology of the present invention has following characteristics:

(1) traditional freezing preservation technology of fish embryo is a programme-control falling temperature method at a slow speed, and this method can't be avoided forming ice crystal in the embryonic cell, thereby fails all the time to succeed in the freezing preservation of fish embryo.The technology of the present invention adopts unique vetrifying solution prescription and technological process, the rate of temperature fall of the vitrifying freeze process of setting up is high, can reduce the formation of ice crystal in the embryonic cell in the temperature-fall period to greatest extent, make the embryo avoid the damage of ice crystal, thereby obtain the embryo and the fry of freezing resurrection.

(2) the technology of the present invention can repeat to obtain the flounder embryo of freezing resurrection, and utilizes the programme-control falling temperature method just to obtain the embryo of once bringing back to life once in a while in the past on freshwater fish, and experimental result can not repeat.The technology of the present invention is preserved in the experiment at 8 glass freezings and was all obtained the freezing resurrection embryo of lefteye flounder that quantity does not wait, from 292 flounder embryo of freezing preservation, obtain to bring back to life 20 of embryos altogether, the minimum resurrection rate of embryo is 1.64% behind the freeze-thaw, the highest resurrection rate is 32.35%, and on average resurrection rate of frozen embryo reaches 6.8%.

(3) make freezing resurrection embryo continue to grow and hatch fry, this result occupies the leading level in the world for obtaining first both at home and abroad with using the technology of the present invention many successes.We preserve in the experiment at 5 glass freezings, all obtain the result that freezing resurrection embryo continues to grow and hatch fry.In 20 pieces of freezing resurrection embryos, have 14 pieces and freeze embryo and hatch fry, incubation rate is 70%.The fry that freezing resurrection embryo hatches, idiosome is transparent, and form is normal, does not have deformity, and is just the same with not freezing normal fry.Prove that fully the technology of the present invention has reliability and repeatability.

(4) the technology of the present invention need not to use expensive instrument and equipment, operate comparatively simply, with low cost, is easy to popularization.

Embodiment:

Its technology path of fish embryo vitrification freezing and storing method is: the thawing → embryo's wash-out and balance → embryo's cultivation of the freezing preservation → straw of the balance of the straw that seals, contains the embryo of the transfer of the balance → embryo before the preparation → embryo cryopreservation of the determining of embryo collection → embryo quality and developmental stage → screening vitrifying basal liquid and antifreeze → vetrifying solution in vetrifying solution in straw, straw → in liquid nitrogen.

The glass freezing that carries out with the flounder embryo successful experiment of preserving now is described below:

(1) embryo collection: carry out the lefteye flounder yielding ability according to routine techniques and grow seedlings, the lefteye flounder fertilized egg of collecting natural spawning under the artificial condition is 14-16 ℃ of cultivation, with the microscopic examination embryonic development stage, choose grow to different times embryos such as muscle segment apparition, tail bud phase, heart beat period, membrane early stage as freezing preservation material.

(2) screening of embryo vitrifying base soln: fish embryo all is to grow in certain water environment.The seawater fish embryo grows different fully with freshwater fish with the environment of life with fry.Therefore, must be according to seawater fish embryo's physiological situation, the base soln that preparation adapts.We select NaCl, KCl, CaCl for use on the basis of a large amount of experiments ₂.2H2O, MgCl ₂.6H ₂O, NaHCO ₃Deng the solution that is mixed with 4 kinds of variable concentrations, under room temperature (15-16 ℃), lefteye flounder embryo's gastrul stage is cultivated, it is grown to membrane, add up its incubation rate.Be chosen to the base soln (be abbreviated as BS) of the highest solution of motility rate as formulate glass liquid and eluent.It the results are shown in Table 1:

Table 1: different basal liquid prescription The selection result

The base soln title	Basal liquid composition (%)						Embryonic hatching (%)
								NaCl	KCl	CaCl 2 .2H 2O	MgCl 2. 6H 2O	NaHCO 3	Total salinity (%)
	BS1 BS2 BS3 BS4	2.375 2.472 2.572 2.792	0.076 0.086 0.086 0.11	0.136 0.146 0.236 0.23	0.466 0.486 0.566 0.64	0.018 0.019 0.028 0.03								3.068 3.209 3.488 3.802	78.33±16.37 81.5±5.57 74.84±6.01 70.73±10.17

By table 1 as seen, the embryonic hatching the highest (81.5%) that base soln BS2 produces is so select the base soln of BS2 as formulate glass liquid and eluent for use.

(3) comparison of antifreeze toxicity: the variety classes antifreeze is not quite similar to the toxicity of different fish embryos, and we are material with the turbot embryo, have carried out the antifreeze screening experiment.The turbot neurula is placed on methyl-sulfoxide (DMSO), dimethyl formamide (DMF), glycerine, ethylene glycol (EG) respectively, 1, Balance Treatment 70min in 2-propane diols (PG) and the methyl alcohol (MeOH) observes embryo's growth then, is counted as motility rate.It the results are shown in table 2.

The comparison of table 2. variety classes antifreeze toxicity

	DMSO DMF glycerine EG PG methyl alcohol
		Embryo number (grain) (n=3)	10 13 23 14 13 12 11 22 15 19 14 11 14 13 12 18 28 17
Survive the embryo number	0 0 0 0 0 0 0 0 0 0 0 0 9 4 12 4 0 6
		Survival rate % average survival %	0 0 0 0 0 0 0 0 0 0 0 0 64.4 30.8 100 22.2 0 35.2 0 0 0 0 65.1 19.2

The result shows to have only the embryo survival in PG (1, the 2-propane diols) and the methyl alcohol, and survival rate is respectively 65.1% and 19.2%, and the embryo in other antifreeze is all dead, and the toxicity of visible PG is minimum, is methyl alcohol secondly.So with 1,2-propane diols (PG) and methyl alcohol (MeOH) are elected to be the antifreeze that the seawater fish embryo cryopreservation is preserved.

(4) screening of suitable vitrified antifreeze: can form the quality of vitrifying and vitrifying state, be one of key of setting up the glass freezing technology.Utilize 1,2-propane diols (PG), methyl alcohol (MeOH), glycerine (Gly), dimethyl formamide (DMF), ethylene glycol (EG) and dimethyl sulfoxide (DMSO) 6 kinds of antifreezes such as (DMSO), PG with the toxicity minimum is a main gene, on four concentration gradient levels of 15,20,25,30% (v/v), make up, utilize BS2 to be base soln, be mixed with the antifreeze solution of A, B, four groups 80 kinds different compositions of C, D, variable concentrations.Above-mentioned various antifreeze solution are filled in straw, be placed on freezing 10-30min in the liquid nitrogen (196 ℃), in 40 ℃ of water-baths, thaw rapidly then, observe vitrifying state freezing and when thawing simultaneously, vitrifying is designated as "+", non-glassization or deglass are designated as "-", experiment repeats 3 times, selects freezing and when thawing the maximum antifreeze of vitrifying number of times be used for vetrifying solution and prepare.The vitrifying degree that now filters out preferably several solns the results are shown in table 3:

Table 3 vetrifying solution is formed and thaw point

The vetrifying solution code name	Form	Total concentration (%v/v)	The vitrifying degree *Freeze-thaw	Easy vitrifying thaw point (℃)
					VSD 1 VSD 2 VSD 5 VSD 9 VSD 10 VSD 14	PG 4+MeOH 1 PG 4+MeOH 2 PG 4+Gly 1 PG 4+DMF 1 PG 4+DMF 2 PG 4+EG 2	45 50 45 45 50 50	3+ 2+，1- 3+ 2+，1- 3+ 2+，1- 3+ 2+，1- 2+，1- 2+，1- 3+ 2+，1-	37～38 35～42 42 39 39～40 35～37

Annotate: 3 vitrifyings of " 3+ " expression when freezing, 2 vitrifyings of " 2+, 1-" expression when thawing, 1 deglass.

As can be seen from Table 3, the vitrifying probability of above-mentioned several vitrification solutions is higher, and the vitrifying degree is better, but they keep the desired temperature of vitrifying state to be not quite similar when thawing.The vitrifying state of some vetrifying solution is pretty good, but their desired thaw point was not low (as VSD1 and VSD14), is exactly temperature range too narrow and small (as VSD5 and VSD9).And have only the vitrifying state of VSD2 good, can in 35-42 ℃ broad water temperature range, thaw again and keep glassy state.

Therefore, take all factors into consideration from aspects such as the toxicity of antifreeze and vitrifying character thereof, we filter out the suitable desirable antifreeze that carries out glass freezing is 1,2-propane diols (PG) and methyl alcohol (MeOH).

(5) preparation of flounder embryo vitrification solution and screening: with the base soln BS2 that filters out in the step (1) with antifreeze 1,2-propane diols (being abbreviated as PG) and methyl alcohol (being abbreviated as MeOH) mix by a certain percentage, be mixed with following four kinds of vitrification solutions (FVS1-FVS4): FVS1 contains BS2 67%, PG 20% and MeOH 13%; FVS2 contains BS2 60%, PG 24% and MeOH 16%; FVS3 contains BS255%, PG 27% and MeOH 18%; FVS4 contains BS2 50%, PG 30% and MeOH 20%.Then lefteye flounder muscle effector phase embryo is placed on and carries out Balance Treatment in the above-mentioned vitrification solution.Survival rate, incubation rate and the abnormal rate of embryo after now will handling are listed in table 4.

Table 4: the survival rate (%) of lefteye flounder muscle effect embryo in different glass liquid

Vetrifying solution	FVS1	FVS2	FVS3	FVS4
					Survival rate of embryo (%) incubation rate (%) abnormal rate (%)	54.29±4.46 86.49±8.78 3.25±5.29	38.68±12.20 87.87±5.24 9.63±4.46	37.8±1.51 69.21±14.1 3.70±6.41	27.71±1.29 39.42±5.43 35.71±12.37

By table 4 as seen, the survival rate after the Balance Treatment and incubation rate are all apparently higher than the FVS4 processed group in vetrifying solution FVS1, FVS2 and FVS3 for the embryo, and abnormal rate then is starkly lower than the FVS4 processed group.Show that thus vetrifying solution FVS1, FVS2 and FVS3 are suitable for the glass freezing of flounder embryo and preserve.

(6) determining of the embryonic development period of suitable vitrifying processing: embryonic development difference in period, to the tolerance difference of antifreeze and vetrifying solution.At the embryo collection in (1) bar, this is divided five steppings to go among the vetrifying solution FVS3 somite embrasure, 16-18 to somite embrasure, tail bud phase embryo and heart beat period embryo the lefteye flounder 4-5, handle 30min, 40min and 50min respectively, use 0.125mol/L sucrose liquid wash-out 10min then.Add the sterilization seawater and cultivate the regular hour, the statistics survival rate, result such as table 5:

Table 5: the survival rate after the different development stage flounder embryo is handled in vetrifying solution FVS3

Embryonic development period	Equilibration time min
				30	40	50
	4～5 pairs of muscle segment embryos	83.8±7.8	74.68±9.56				63.96±9.25
16～18 pairs of muscle segment embryos				81.04±2.89	78.13±5.25	69.41±10.77
	The tail bud embryo	81.72±9.58	67.47±7.99				56.37±12.79
Embryo heart rate				14.42±9.46	12.2±6.68	4.41±1.85

As can be seen from Table 5,4-5 is higher to muscle segment embryo and tail bud phase embryo survival rate behind the Balance Treatment different time in vetrifying solution FVS3 to muscle segment embryo, 16-18, and the heart beat period embryo is then obviously low to the adaptive capacity of FVS3.Show that thus 4-5 is suitable for the vetrifying solution processing to muscle segment embryo, 16-18 to muscle segment embryo and tail bud phase embryo.

(7) determining of embryo's equilibrium temperature: respectively under 15 ℃ and 4 ℃, utilize the FVS3 five-step approach to handle the lefteye flounder tail bud and begin to form phase embryo 40min, without freezing, directly use the sucrose wash-out 10min of 0.125mol/L, cultivate (15.5 ℃) with filtering sea, add up its survival rate, more different equilibrium temperatures the results are shown in Table 6 to the influence of survival rate of embryo.By table 6 as seen, flounder embryo apparently higher than the survival rate under 4 ℃, is an active balance temperature so select 15 ℃ in the survival rate after 15 ℃ of balances.

Table 6: the survival rate of tail bud embryo (%) under the different equilibrium temperatures

Equilibrium temperature ℃	15.0	4.0
			Five step equilibration time min elution time min survival rates (%)	40 10 83.67±5.76	40 10 66.27±14.47

(8) embryo's the determining of equilibrium step in vetrifying solution: adopt one-step method, three-step approach or five-step approach that muscle segment apparition embryo was progressively carried out the transition in the vetrifying solution (20%DMSO and 25% dimethyl formamide) at room temperature Balance Treatment 40 minutes, back 1mol/L sucrose liquid wash-out, cultivate with 12 ℃ of filtering seas again, observe its survival rate and incubation rate; The result is as shown in table 7:

Table 7: the survival rate of embryo under different balance methods

Balance method	One-step method	Three-step approach	Five-step approach
				Equilibration time (min)	40	40	40
The average incubation rate (%) of average survival (%)	0 0	4.3 22.2	28.3 36.3

By table 7 as seen, through 3 repeated tests, therefore survival rate after the muscle segment phase embryo employing five-step approach balance and incubation rate, in freezing preservation experiment subsequently, all adopt five step balancing methods apparently higher than three-step approach and one-step method group.

(9) glass freezing: will be in vetrifying solution the good embryo of balance, suck straw together with vetrifying solution, the straw diameter is 2.5mm, length is 10cm.Every pipe sucks 250 μ l vetrifying solutions, wherein surplus the embryo 10 piece, the embryo is evenly distributed in straw, avoids flocking together; After sealing with fire, preserve carrying out glass freezing in the liquid nitrogen of straw with the above rate of temperature fall input-196 of 2000 ℃/min ℃.Holding time length is determined according to actual needs.

(10) screening of eluent: adopt five-step approach that tail bud phase embryo is placed on Balance Treatment 30min in the VSD2 vetrifying solution, then at-20 ℃ of freezing 10min, respectively with 0.0625,0.125,0.25,0.5 or the 1.0Mol/L sucrose solution carry out wash-out, behind the 10min embryo is transferred in the seawater, at room temperature (15 ℃) cultivate certain hour, the statistics survival rate.As shown in table 8.

By table 8 as seen, the elute effect of 0.125Mol/L sucrose liquid is best, and survival rate of embryo is the highest.

Table 8: the elute effect of variable concentrations sucrose liquid

Sucrose concentration (M)	0.0625	0.125	0.25	0.5	1.0
						Total sample number survives embryo average survival (%)	72 5 4.76	85 14 18.8	77 6 8.67	86 7 9.83	105 7 5.7

(11) embryo thawing and cultivation: straw is taken out from liquid nitrogen, insert fast in the 39-40 ℃ of water-bath, shaking of not stopping thawed, and straw is moved out to time of entering water-bath in 1-2 second from liquid nitrogen, and overlong time can devitrification, causes embryo's fragmentation.Glassy state is milky white by transparent change in water-bath, bleach again, when embryo's milky is about to disappear straw is taken out from water-bath, cut off and seal, sucrose eluent with 2ml0.125M is flushed to wash-out in the culture dish rapidly, add 14-16 ℃ of sterilization seawater behind the wash-out 10min and cultivate, up to the fry hatching membrane.That now the flounder embryo glass freezing is preserved mainly the results are shown in table 9.

Table 9: flounder embryo glass freezing saving result

Vitrification solution	Embryonic development period	Cooling time	Sample number	Bring back to life the embryo number	Resurrection rate (%)	Hatch the fry number	Become live time (h)
								FVS3 FVS1 FVS3 FVS3 FVS4 FVS4 FVS2 FVS3 amounts to	Embryo before the 14 somite embrasure tail bud embryo tail bud embryo tail bud embryo tail bud embryo tail bud embryo muscle effects phase embryo membranes	1h 1h 7h33min 1h22min 1h8min 2h20min 1h31min 2h34min	21 34 35 32 44 35 30 61	1 11 2 1 1 1 2 1 20	4.76 32.35 5.71 3.13 2.27 2.86 6.67 1.64	1 9 1 0 0 0 2 1 14	108 90 67 18 72 61 62 14

By table 9 as seen, use technique scheme, to lefteye flounder body segment phase embryo, the tail bud embryo, muscle effect embryo, membrane embryo in early stage has carried out glass freezing and has preserved experiment, preserves in the experiment at 8 different glass freezings, successfully obtains freezing resurrection embryo.From 292 pieces of frozen embryo, obtain 20 pieces of embryos of bringing back to life fully altogether, freeze embryo resurrection rate and reach as high as 32%, the back embryo morphology that thaws is normal, continue to grow and hatch fry, wherein 14 pieces bring back to life embryos and successfully hatch fry, bring back to life percentage that the embryo hatches fry up to 70%, and it is normal to freeze the fry form that embryo hatches.

Claims (1)

1. a fish embryo vitrification freezing and storing method is characterized in that its operating technology is: comprise Balance Treatment, freezing preservation and the thaw resurrection of embryo in liquid nitrogen before fish embryo is collected, prepared basal liquid and screens antifreeze, formulate glass liquid, embryo cryopreservation; The collection of its fish embryo: be early stage in body segment apparition-membrane; The vetrifying solution preparation: be the mixed liquor of basal liquid and antifreeze, basal liquid (BS2) is: NaCl2.472%, KCl0.086%, CaCl ₂.2H ₂O0.146%, MgCl ₂.6H ₂O0.486%, NaHCO ₃0.019%; Antifreeze is: 1, and 2-propane diols (PG), methyl alcohol (MeOH); The vetrifying solution prescription is: BS2:50-67%, PG:20-30%, MeOH:13-20%; Balance Treatment before the embryo cryopreservation: the embryo divided in concentration is 25%, 33%, 50%, 67% and 100% vetrifying solution step by step successively carry out Balance Treatment five times, handle 45-55min; Per step 9-11min; Freezing preservation in the liquid nitrogen: the embryo 9-13 grain that balance is good is drawn in diameter 3mm, the long 10cm straw together with the vetrifying solution of 250 μ l, after the hot-press sealed, puts into quick-frozen preservation in-196 ℃ of liquid nitrogen with the rate of temperature fall of 2000 ℃/min; Thaw and cultivate: straw is shifted out from liquid nitrogen, insert in 39-40 ℃ of water-bath fast in the time in second at 1-2 it is thawed rapidly, when embryo's milky is about to disappear, cut off straw, with the sucrose eluent of 2ml 0.125M, the embryo to be poured carry out wash-out in the vessel, the time is 10min; Adding 14-16 ℃ of sterilization seawater afterwards cultivates.