CN104521943A - Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen - Google Patents
Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen Download PDFInfo
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Abstract
The invention discloses an ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen. Concretely, the method comprises an ultralow-temperature preservation method for oxyeleotris marmorata semen and a recovery method for ultralow-temperature preserved oxyeleotris marmorata semen. The ultralow-temperature preservation method for oxyeleotris marmorata semen comprises the following steps: (1) acquiring oxyeleotris marmorata semen; (2) detecting semen activity; (3) diluting semen; (4) performing pre-refrigeration balancing on the semen liquid; (5) adding an antifreeze agent and subpackaging; (6) performing programmed cooling; and (7) putting in liquid nitrogen. The recovery method for ultralow-temperature preserved oxyeleotris marmorata semen comprises: (1) taking out frozen semen from the liquid nitrogen tank; (2) unfreezing; (3) storing frozen semen for usage; and (4) performing artificial insemination. The method performs ultralow-temperature preservation and recovery on oxyeleotris marmorata semen, thereby providing high-quality semen fluid for crossbreeding, artificial breeding and germplasm preservation of oxyeleotris marmorata, and laying a foundation for heredity improvement of oxyeleotris marmorata. The method is simple and rapid in operation, low in cost and high in practicability.
Description
Technical field
The invention belongs to Aquaculture Science field, relate to fish germplasm Techniques of preserving, be specifically related to a kind of Oxyeleotris marmoratus sperm super-low temperature freezing and preserve and method for resuscitation.
Background technology
At present, fish germplasm is preserved main based on Semen routine.The preservation of sperm can be divided into normal temperature preservation, Cord blood and Ultra-cryofreezing preservation three kinds.The life-span of sperm terminates with the approach exhaustion of its energy.Its energy ezpenditure is mainly in two: one is motion (wagging the tail travelling); Second is regulate intraor extracellular osmotic balance (non-active transportation energy consumption).Therefore, slow down the energy ezpenditure of sperm, just can by it life.It is physiological property according to sperm that normal temperature is preserved, and prepares certain density dilution, thus reduces sperm and be used for energy ezpenditure when regulating osmotic balance and then extend life-span of sperm, usually can extend dozens of minutes to several hours.The principle of Cord blood is: low temperature can reduce sperm metabolism activity (reduction of low temperature enzyme efficiency), usually can extend several days to tens days.Excised Embryos is then allow sperm under ultralow temperature (-196 DEG C) condition, and its motion and metabolism almost stop completely, and sperm is in class torpor, and life is to preserve close to inactive state.Sperm can indefinitely be preserved in the ideal situation, can " bring back to life " again after thawing.
Oxyeleotris marmoratus (Oxyeleotrismarmorata) is that China introduces a fine variety and the famous and precious fresh-water fishes kind cultivated, and because its bodily form is like bamboo shoot shell, therefore popular name is bamboo shoot shell fish.This fish originates in Southeast Asia Thailand one band, maximum individuality can reach 5 ~ 6 kilograms, its fine and tender taste, be of high nutritive value, delicious flavour and thorn is few, a kind of famous and precious food fish, Southeast Asian countries and Hongkong and Macro, Taiwan etc. area and Japan very popular, be the main freshwater fish products of the large outlet of these countries in Southeast Asia.Rely on natural resources to catch in the level of cultivation because state of origin bamboo shoot shell fish still rests on, the consumption demand that market is powerful cannot be met, cause this marketable fish wholesale market price at present to increase to 160 ~ 200 yuan/kg.As can be seen here, the market prospects of Oxyeleotris marmoratus are good, are a kind of aquaculture new varieties of emerging, Peak output, high benefit.In order to the breeding of new variety of Oxyeleotris marmoratus, artificial propagation and preserving seed can be carried out further, for market provides Oxyeleotris marmoratus new varieties and the seed of high-quality more, need to carry out the preservation of Oxyeleotris marmoratus sperm super-low temperature and artificial propagation.But, it is no matter the Excised Embryos of sperm, still carry out artificial propagation, all need a kind of dilution to the fresh row dilution that progresses greatly of Oxyeleotris marmoratus, and need a kind of activation solution to fresh essence or freeze essence (sperm after Excised Embryos thaws again) and activate.
In Oxyeleotris marmoratus seminal fluid, the density of contained sperm is very high, generally can reach 1.8 × 10
9individual/more than mL, when carrying out Excised Embryos to Oxyeleotris marmoratus sperm, smart stoste containing high density sperm directly can not carry out Ultra-cryofreezing preservation, this is because in refrigerating process, when temperature drop to 0 DEG C ~-20 DEG C interval time, ice crystal can be formed in the seminal fluid be saved, ice crystal stabs the sperm of surrounding as sharp cutter, sperm is punctured, sperm concentration in seminal fluid is higher, the probability that sperm is punctured is also higher, this failure that ice crystal will directly cause sperm super-low temperature freezing to be preserved to the physical damnification of sperm in refrigerating process.Therefore, must dilute sperm before freezing.And when carrying out Oxyeleotris marmoratus artificial propagation, due to very high containing sperm concentration in seminal fluid, if do not dilute with regard to use, be not only a kind of waste to this milt liquid, and fresh smart stickiness is large, sperm is not easily mixed with ovum, thus causes Insemination effect also bad.If after diluting seminal fluid, then be used for carrying out artificial insemination, can greatly reduce test-tube cost, good Insemination effect can also be played simultaneously, obtain higher fertilization rate.Therefore, the effect of Oxyeleotris marmoratus spermatozoa diluent is exactly under the prerequisite not activating sperm, dilutes sperm, makes sperm keep its original biological property as much as possible.
When sperm is by Excised Embryos, in order to improve the freezing tolerance of sperm, except diluting seminal fluid, also need to add a certain amount of antifreeze, as methyl-sulfoxide (DMSO) etc. in the seminal fluid of dilution.Because antifreeze has very strong water imbibition, when being added to after in seminal fluid, a part of water in spermoblast can be made to be sucked out.The water loss of spermoblast causes the cell sap concentration in spermoblast to raise, and the freezing point of sperm also increases, thus improves the freezing tolerance of sperm.Also raise just because of the cell sap concentration in spermoblast, the osmotic pressure of sperm also can increase.Like this, the osmotic pressure freezing essence can exceed 30 ~ 40% compared with fresh essence.Freeze essence containing high internal penetration pressure like this, carry out artificial insemination if directly activated with fresh water, under huge permeable pressure head effect, it freezes single-mindedly meets water and just may break, thus makes artificial insemination unsuccessfully.The way addressed this problem is exactly carry out alternative fresh water with the activation of spermatozoa liquid with certain osmotic pressure to freeze to Oxyeleotris marmoratus the line activating that progresses greatly, to reduce this permeable pressure head.Therefore, the effect of Oxyeleotris marmoratus activation of spermatozoa liquid is exactly reduce this permeable pressure head, making Oxyeleotris marmoratus freeze essence and can not break when activating, realizing the artificial insemination with freezing this fish of row of progressing greatly smoothly.In addition, activation of spermatozoa liquid is except reducing the permeable pressure head frozen between essence and activation solution, and protection is frozen outside smart effect of not breaking in activation, also has to the fresh essence of general Oxyeleotris marmoratus the effect that sperm viability is optimized.This is because activation solution is made up of different ions or macromolecular substances, the motion of these ions and macromolecular complex confrontation sperm can play certain optimization function.
At present, the species extinction caused due to environmental deterioration, inbreeding etc., plant the phenomenons such as matter is degenerated, quality declines and disease takes place frequently and be on the rise, the effective tool that Ultra-cryofreezing preservation technology is preserved for a long time as gene becomes the first-selection addressed these problems gradually.In addition, Excised Embryos technology can also constantly provide sperm material for a long time for genetic breeding research, and this is at female androgenesis, sexual control and produce using sperm as carrier in genetically modified organism etc. and all have using value.
So far the preservation of Eleotridae fish sperm and method for resuscitation are reported seldom, have no the report to the preservation of Oxyeleotris marmoratus sperm super-low temperature freezing and method for resuscitation.Because Oxyeleotris marmoratus belongs to tropic fishes, its original ancestry is from ocean, migrated fresh water life afterwards, its sperm storage and method for resuscitation be both different from freshwater fish and be also different from seawater fish, according to published freshwater fish or seawater fish sperm storage and method for resuscitation Oxyeleotris marmoratus sperm super-low temperature freezing preserved and recovery all unsuccessful; Await further research.
Summary of the invention
Oxyeleotris marmoratus sperm super-low temperature freezing is preserved and the summary of the invention of method for resuscitation comprises the preservation of Oxyeleotris marmoratus sperm super-low temperature and a method for resuscitation two large divisions, and its concrete summary of the invention is as follows:
The method that Oxyeleotris marmoratus sperm super-low temperature is preserved, the steps include:
(1) collection of Oxyeleotris marmoratus sperm: select sexual gland fully-developed Oxyeleotris marmoratus milter, dries around gonopore with dry towel and the water of belly.Gently extrude milter belly with have gentle hands, after extruding seminal fluid, draw seminal fluid with the dropper of drying, beginning 3-5 drips seminal fluid and does not gather, and is then clamp-oned by seminal fluid in dry container, mixing.
(2) sperm viability detects: under the microscope of 10 × 20 times, carry out microscopy, the average motility rate of the fresh essence of computer aided detection reaches more than 90%, averaged curve movement velocity (VCL) reaches more than 35 micro-meter per seconds, mean linear movement velocity reaches more than 20 micro-meter per seconds, and this sperm just can be used for Ultra-cryofreezing preservation.Sperm qualified for microscopy is put into 4 DEG C of Refrigerator stores for subsequent use.
(3) dilution of sperm: accurately measure the Oxyeleotris marmoratus seminal fluid needing to be frozen, add seminal fluid about 3 times of volumes (in order to packing is convenient, the amount of dilution can suitably increase and decrease a bit, increase and decrease amount be generally no more than 10% of total amount) spermatozoa diluent, make the thinner ratio of seminal fluid and dilution reach about 1: 3.Seminal fluid and dilution mix, for subsequent use.
Described spermatozoa diluent is the distinctive spermatozoa diluent of Oxyeleotris marmoratus, and its formula is: 7.0g/L sodium chloride (NaCl), 1g/L potassium chloride (KCl), 15g/L glucose (C
6h
12o
6).This dilution salinity 7.3, pH is 7.5.Be placed on 4 DEG C of Refrigerator stores, for subsequent use.
(4) seminal fluid freeze forward horizontal stand: by dilution after seminal fluid put into 4 DEG C of refrigerators, carry out freezing forward horizontal stand.The forward horizontal stand time of freezing of Oxyeleotris marmoratus seminal fluid is 15 ~ 45 minutes.Within 15 minutes, be minimum equilibration time, if equilibration time was less than 15 minutes, then refrigerating effect can decline.Within 45 minutes, be the longest equilibration time, within 15 minutes to 45 minutes, refrigerating effect does not have difference, and more than 45 minutes, then refrigerating effect reduced gradually along with the prolongation of time.
(5) with antifreeze and packing: directly add antifreeze in the Oxyeleotris marmoratus seminal fluid balanced, the final concentration of antifreeze in seminal fluid is made to reach 10%.After antifreeze adds, mix immediately, divide and be filled to 0.5mL straw, 6 straws load in 1 straw cover, 1 freezing frame can be put 2 straw covers.Note can not leaving bubble in all pipes, a point process of assembling must complete in ice face, and the pipe that packing completes, before not entering programmed cooling instrument, should all be embedded in ice, guarantees that the seminal fluid in pipe can not heat up.
Described antifreeze is methyl-sulfoxide (DMSO), without the need to preparation, directly uses stoste.Antifreeze utilizes its water sorption, and the part water in sperm is deviate from, and cytoplasm concentration raises, thus reach the object reducing sperm freezing point, prevents sperm sperm inside in refrigerating process from forming ice crystal.Be placed on 4 DEG C of Refrigerator stores, for subsequent use.
(6) programmed cooling: cooling carries out in programmed cooling instrument, and programmed cooling instrument is controlled by calculator, after calculator is transfused to the cooling process of designed, designed, programmed cooling instrument can start cooling according to the program designed.Point seminal fluid installed is put into the refrigerating chamber of programmed cooling instrument with the fastest speed.The cooling process of Oxyeleotris marmoratus, from 0 DEG C, is down to-15 DEG C with the speed of 5 DEG C/min, stops 5 minutes, and then be down to-85 DEG C with 25 DEG C/min of speed from-15 DEG C at-15 DEG C, finally locates balances 10 minutes at-85 DEG C.
Described cooling process is: from 0 DEG C, is down to-15 DEG C with the speed of 5 DEG C/min, stops 5 minutes, and then be down to-85 DEG C with 25 DEG C/min of speed from-15 DEG C at-15 DEG C, finally locates balances 10 minutes at-85 DEG C.
(7) enter liquid nitrogen: after having lowered the temperature, directly straw or cryovial that seminal fluid is housed are taken out together with freezing support from programmed cooling instrument, put into liquid nitrogen container immediately, preserve in-196 DEG C of temperature.
The method for resuscitation that Oxyeleotris marmoratus sperm super-low temperature is preserved, the steps include:
(1) take out from liquid nitrogen container and freeze essence: first liquid nitrogen surface balance 15 minutes, that then takes off 1 straw cover with long forceps freezing support from liquid nitrogen container freezes essence (1 in-built 6 straw of straw cover, often prop up straw 0.5mL is housed freezes essence, 3mL freezes essence altogether).After liquid nitrogen container mouth places 30 ~ 60 seconds, from straw cover, take out straw, prepare to thaw.
(2) thaw: straw is directly put into 30 DEG C of water-baths, constantly rock straw in a water bath, when seeing after 10 seconds that in straw, bubble can move up and down, namely illustrate that freezing essence in straw thaws.
(3) freezing essence deposits with for subsequent use: focused on by the seminal fluid after thawing in a dry container, put into 4 DEG C of refrigerators for subsequent use.The refrigerator resting period is no more than at most 30 minutes and just must be used for artificial insemination.
(4) artificial insemination: the 3mL for subsequent use that thaws is frozen essence and pours in the ovum extruded, stir evenly gently with feather.Then add the general activation solution of sperm that 50mL prepares in advance, gently stir 3 minutes, leave standstill after 1 minute, then add 500mL with supernatant water activate sperm to reach fertilization, ovum can be moved in incubator and hatch.
Described activation of spermatozoa liquid is the general activation solution of Oxyeleotris marmoratus, and its formula is: 2.6g/L sodium chloride (NaCl), 0.4g/L potassium chloride (KCl), 2.4g/L Tris-HCl.This activation solution pH is 8.2.Be placed on 4 DEG C of Refrigerator stores, for subsequent use.
Embodiment
Instantiation of the present invention is as follows, and design parameter, for illustrating needed for technical solution of the present invention, should not be construed as limiting the scope of the invention.
One, the Ultra-cryofreezing preservation of Oxyeleotris marmoratus sperm
1, spermatozoa diluent is equipped with: fill a prescription as 7.0g/L sodium chloride (NaCl), 1g/L potassium chloride (KCl), 15g/L glucose (C
6h
12o
6).This dilution salinity is 7.3, pH is 7.5.Be placed on 4 DEG C of Refrigerator stores, for subsequent use.
2, seminal fluid prepares: select body weight more than 500 grams and the Oxyeleotris marmoratus milter of gonadal maturation, dries around gonopore and the water of belly with dry towel.Gently extrude milter belly with have gentle hands, after extruding seminal fluid, draw seminal fluid with the dropper of drying, beginning 3-5 drips seminal fluid and does not gather, and is then clamp-oned by seminal fluid in dry container, mixing.After seminal fluid is activated with clear water, microscopy is carried out under the microscope of 10 × 20 times, the average motility rate of the fresh essence of computer aided detection reaches more than 90%, and averaged curve movement velocity (VCL) reaches more than 35 micro-meter per seconds, and mean linear movement velocity reaches more than 20 micro-meter per seconds.
3, Ultra-cryofreezing preservation is prepared: accurately measure Oxyeleotris marmoratus 0.75mL × 6 seminal fluid needing to be frozen, add 2.25mL × 6 spermatozoa diluent, mixing.Seminal fluid after dilution is put into 4 DEG C of refrigerators carry out freezing forward horizontal stand, add after 30 minutes as antifreeze methyl-sulfoxide (DMSO), make the final concentration of antifreeze in seminal fluid reach 10%.
4, programmed cooling: after antifreeze adds, mixes immediately, divides and is filled to 0.5mL straw, and 6 straws load in 1 straw cover, 1 freezing frame can be put 2 straw covers.Point seminal fluid installed is put into the refrigerating chamber of programmed cooling instrument with the fastest speed.Described cooling process is: from 0 DEG C, is down to-15 DEG C with the speed of 5 DEG C/min, stops 5 minutes, and then be down to-85 DEG C with 25 DEG C/min of speed from-15 DEG C at-15 DEG C, finally locates balances 10 minutes at-85 DEG C.After having lowered the temperature, directly straw or cryovial that seminal fluid is housed are taken out together with freezing support from programmed cooling instrument, put into liquid nitrogen container immediately, preserve in-196 DEG C of temperature.
Two, the recovery of Oxyeleotris marmoratus Excised Embryos sperm:
1, activation of spermatozoa liquid is equipped with: its formula is: 2.6g/L sodium chloride (NaCl), 0.4g/L potassium chloride (KCl), 2.4g/L Tris-HCl.This activation solution pH is 8.2.Be placed on 4 DEG C of Refrigerator stores, for subsequent use.
2, squeeze ovum: the Oxyeleotris marmoratus raun of body weight more than 400 grams is hastened parturition, after reaching effector phase, adopt belly extrusion to be clamp-oned gently in little ceramic bowl by ovum.
3, thaw and recover: the Oxyeleotris marmoratus taking out freezing more than 1 month from liquid nitrogen container freezes essence: first liquid nitrogen surface balance 15 minutes, that then takes off 1 straw cover with long forceps freezing support from liquid nitrogen container freezes essence (1 in-built 6 straw of straw cover, often prop up straw 0.5mL is housed freezes essence, 3mL freezes essence altogether).After liquid nitrogen container mouth places 30 ~ 60 seconds, from straw cover, take out straw, straw is directly put into 37 DEG C of water-baths, constantly rock straw in a water bath, when seeing after 10 seconds that in straw, bubble can move up and down, namely illustrate that freezing essence in straw thaws.
4, artificial insemination: the 3mL for subsequent use that thaws is frozen essence and pour in the ovum of the cloud plate Oxyeleotris marmoratus of extruding, stir evenly gently with feather.Then the general activation solution of sperm that 50mL prepares in advance is added, stir 3 minutes gently, leave standstill after 1 minute, add 500mL again and activate sperm to reach fertilization with supernatant water, fertilized egg can be moved in incubator and hatch, fertilization rate more than 85%, utilize simultaneously fraction recover after freeze the row microscopy that progresses greatly, average viability reaches more than 90%.
Claims (8)
1. a method for Oxyeleotris marmoratus sperm super-low temperature preservation, the steps include:
(1) collection of Oxyeleotris marmoratus sperm: select sexual gland fully-developed Oxyeleotris marmoratus milter, dries around gonopore with dry towel and the water of belly; Gently extrude milter belly with have gentle hands, after extruding seminal fluid, draw seminal fluid with the dropper of drying, beginning 3-5 drips seminal fluid not, is then clamp-oned by seminal fluid in dry container, mixing;
(2) sperm viability detects: under the microscope of 10 × 20 times, carry out microscopy, the average motility rate of the fresh essence of computer aided detection reaches more than 90%, averaged curve movement velocity (VCL) reaches more than 35 micro-meter per seconds, mean linear movement velocity reaches more than 20 micro-meter per seconds, and this sperm just can be used for Ultra-cryofreezing preservation; Sperm qualified for microscopy is put into 4 DEG C of Refrigerator stores for subsequent use;
(3) dilution of sperm: accurately measure the Oxyeleotris marmoratus seminal fluid needing to be frozen, add the spermatozoa diluent of seminal fluid 3 times of volumes, make the thinner ratio of seminal fluid and dilution reach 1: 3; Seminal fluid and dilution mix, for subsequent use;
(4) seminal fluid freeze forward horizontal stand: by dilution after seminal fluid put into 4 DEG C of refrigerators, carry out freezing forward horizontal stand; The forward horizontal stand time of freezing of Oxyeleotris marmoratus seminal fluid is 15 ~ 45 minutes;
(5) with antifreeze and packing: directly add antifreeze methyl-sulfoxide (DMSO) in the Oxyeleotris marmoratus seminal fluid balanced, the final concentration of antifreeze in seminal fluid is made to reach 10%; After antifreeze adds, mix immediately, divide and be filled to straw; Can not leave bubble in all pipes, a point process of assembling must complete in ice face, and the pipe that packing completes, before not entering programmed cooling instrument, should all be embedded in ice;
(6) programmed cooling: by point seminal fluid installed to put into the refrigerating chamber of programmed cooling instrument fast; The cooling process of Oxyeleotris marmoratus, from 0 DEG C, is down to-15 DEG C with the speed of 5 DEG C/min, stops 5 minutes, and then be down to-85 DEG C with 25 DEG C/min of speed from-15 DEG C at-15 DEG C, finally locates balances 10 minutes at-85 DEG C;
(7) enter liquid nitrogen: after having lowered the temperature, directly straw or cryovial that seminal fluid is housed are taken out, put into liquid nitrogen container immediately, preserve in-196 DEG C of temperature.
2. the method for claim 1, is characterized in that, described step (1) milter used is Oxyeleotris marmoratus (Oxyeleotris marmorata), and body weight is more than 500 grams.
3. the method for claim 1, it is characterized in that, spermatozoa diluent formula described in described step (3) is: 7.0g/L sodium chloride (NaCl), 1g/L potassium chloride (KCl), 15g/L glucose (C6H12O6); This dilution salinity is 7.3, pH is 7.5; Be placed on 4 DEG C of Refrigerator stores, for subsequent use.
4. the method for claim 1, is characterized in that, in described step (4), the forward horizontal stand step of freezing of seminal fluid is: the seminal fluid after dilution is put into 4 DEG C of refrigerators, carries out freezing forward horizontal stand; The forward horizontal stand time of freezing of Oxyeleotris marmoratus seminal fluid is 15 ~ 45 minutes.
5. the method for claim 1, is characterized in that, antifreeze methyl-sulfoxide (DMSO) final concentration in seminal fluid described in described step (5) reaches 10%.
6. the method for claim 1, it is characterized in that, cooling process described in described step (6) is: from 0 DEG C,-15 DEG C are down to the speed of 5 DEG C/min, stop 5 minutes at-15 DEG C, and then be down to-85 DEG C with 25 DEG C/min of speed from-15 DEG C, finally locate balances 10 minutes at-85 DEG C.
7. the method for resuscitation after the preservation of Oxyeleotris marmoratus sperm super-low temperature, is characterized in that, comprise the following steps:
(1) take out from liquid nitrogen container and freeze essence: first liquid nitrogen surface balance 15 minutes, then take off to be equipped with from liquid nitrogen container with long forceps and freeze smart straw; After liquid nitrogen container mouth places 30 ~ 60 seconds, prepare to thaw;
(2) thaw: straw is directly put into 37 DEG C of water-baths, constantly rock straw in a water bath, when seeing after 10 seconds that in straw, bubble can move up and down, namely illustrate that freezing essence in straw thaws;
(3) freezing essence deposits with for subsequent use: focused on by the seminal fluid after thawing in a dry container, put into 4 DEG C of refrigerators for subsequent use; The refrigerator resting period is no more than at most 30 minutes and just must be used for artificial insemination;
(4) artificial insemination: the 3mL for subsequent use that thaws is frozen essence and pours in the ovum extruded, stir evenly gently with feather; Then add the general activation solution of sperm that 50mL prepares in advance, gently stir 3 minutes, leave standstill after 1 minute, then add 500mL with supernatant water activate sperm to reach fertilization, fertilized egg can be moved in incubator and hatch.
8. as right wants the method as described in 7, it is characterized in that, the activation of spermatozoa formula of liquid described in described step (4) is: 2.6g/L sodium chloride (NaCl), 0.4g/L potassium chloride (KCl), 2.4g/L Tris-HCl; This activation solution pH is 8.2, is placed on 4 DEG C of Refrigerator stores, for subsequent use.
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