CN1596670A - Practicalization method for frozen preserving sperm of fish - Google Patents
Practicalization method for frozen preserving sperm of fish Download PDFInfo
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Abstract
A practical method for freeze storage of fish sperm features that the diluent, antifreezing agent and container for the freeze storage of fish sperm chosen from many schemes, and a three-step freezing mode and a quick thawing method are designed.
Description
Technical field:
The invention belongs to the animal sperm superfreeze preservation technology in the cryobiology, is a kind of at the medium-term and long-term practicability technical method of preserving fish sperm of ultralow temperature state (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 the overfishing of fishery resources, unordered utilization etc., 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; learn a skill by low-temperature biological; set up gamete and the embryo cryopreservation storehouse of important cultured fishes original seed and 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 genetic Protection of Diversity and the culture fishery.
Sperm freezing is preserved a kind of method of technology as long preservation animal germplasm, and the aspect has all demonstrated huge application potential preventing that fish species extinction and fish genetic breeding, fish from growing seedlings etc.Although begin from the seventies in the world, carried out the research work that freshwater fish such as salmon trout class, cyprinid fish and some seawater fish freezing of semen are preserved, but sperm freezing preservation technology exist freeze that smart motility rate is not high enough, rate of fertilization is not high, freeze smart reserve capacity little, do not reach problems such as practicability level.Fish sperm low temperature preservation research is also at the experimental stage, and the sperm of preservation is little, is difficult to satisfy the needs of fish production and germplasm preservation.Once do the research that the perch sperm freezing is preserved as (1996) such as domestic big vast Wan Shu, but do not make the frozen effect that sperm is checked in the insemination test.The freezing preservation of fish sperm aspect abroad, Dreanno etc. (1997) had once carried out the research that the turbot sperm freezing is preserved with the 0.25ml straw as frozen container, but the polyspermism rate of freezing that obtains only is 58%, report does not freeze the incubation rate of polyspermism fry, this article reported method reserve capacity is little simultaneously, can't satisfy the requirement that sperm bank makes up.
Summary of the invention:
The objective of the invention is in order to set up the technical method of cyprinid fish and the freezing preservation of important marine fish sperm mass, for the preservation of fish germplasm, genetic diversity protection, genetic breeding and fish production provide the practicability technical method.
The present invention realizes by following technical scheme: the packing that its sperm freezing is preserved dilution that technical method comprises that sperm collection, sperm freezing preserve the selecting for use of the preparation of dilution, antifreeze, sperm and balance, sperm is thawed and is inseminated with freezing, frozen semen.
1, sperm collection: towel off and do ripe milter gonopore zone, gently press belly, collect the seminal fluid of extruding with suction pipe.Place clean bottle.Seminal fluid is put short-term preservation on ice, behind the microscopy survival rate is used for preserving at the seminal fluid more than 80%.
2, sperm freezing is preserved the preparation of dilution:
Fresh water cyprinid fish (as black carp, grass carp, silver carp, flathead, carp, crucian carp, bream etc.) semen diluent is D-15, and its prescription is: NaCl 135-136.75mM, KCl 6-6.71mM and glucose 83-83.33mM;
Seawater fish: the turbot spermatozoa diluent is TS-2, and its prescription is: Tris-Cl 9-11mM, pH 8.0, sucrose 105-115mM, KHCO
395-105mM, 9-11% hyclone (volume ratio).Perch, lefteye flounder and stone flounder spermatozoa diluent are MPRS, and its prescription is: NaCl 59.35-61.35mM, NaH
2PO
41.70-1.90mM, NaHCO
32.5-3.5mM, KCl 4.73-5.73mM mM, CaCl
22H
2O 0.63-1.63mM, MgCl
26H
2O 0.63-1.63mM, D-Glucose 50.55-60.55mM.
3, select antifreeze for use: the freezing preservation antifreeze of above-mentioned fish sperm is DMSO, and its suitable concentration is 8-10%.
4, the dilution of sperm and balance: after the dilution of black carp, grass carp, silver carp, flathead, carp, crucian carp, bream, perch, lefteye flounder and stone flounder seminal fluid and precooling in 4 ℃ of refrigerators mixes in the ratio of (volume ratio) 1: 1 or 1: 2, in 4 ℃ of refrigerator balance 20-30min.Directly freezing after the turbot seminal fluid then dilutes, need not balance period.
5, the packing of sperm and freezing: the semen diluent that balance is good is sub-packed in the frozen pipe of 2-5ml with 1.0-4.5ml, carries out freezing preservation according to three step cooling patterns.I.e. 6 centimeters balance 10min above liquid nitrogen surface, balance 5min on liquid nitrogen surface drops in the liquid nitrogen then and preserves.Best rate of temperature fall is 31 ℃/min (from 16 ℃ to-15 ℃) and 18.6 ℃/min (from-12 ℃ to-180 ℃).
6, thawing and insemination of frozen semen: when thawing, earlier frozen pipe is proposed to place liquid nitrogen steam balance 5 minutes from liquid nitrogen, from liquid nitrogen container, take out then, be placed on quick-thawing in 37 ℃ of water-baths.Freeze and carry out artificial insemination with bright ovum after essence is thawed, way is: frozen semen is fallen on ovum, and stirring and evenly mixing adds a little fresh water (cyprinid fish sperm) or seawater (seawater fish sperm) and activates sperm, makes it and ovum fertilization.Place incubator to hatch embryonated egg after the insemination.
Embodiment:
One, saves as example with seawater fish turbot and perch seminal fluid superfreeze, to of the present invention
Embodiment describes.
1. embodiment is preserved in the turbot freezing of semen
(1) gently is pressed into ripe turbot milter belly, collects the seminal fluid of anuria dirt simultaneously with suction pipe, at the microscopically microscopy.The microscopy method is: dip in toothpick and get a little sperm on slide, nature seawater activates then, and examines under a microscope immediately.The survival rate of sperm is the percentage that the motion sperm accounts for total sperm count in the field of microscope of seminal fluid activation back.Survival rate is used for experiment at the seminal fluid more than 85%.
(2) the turbot sperm after the collection directly is diluted among the TS-2 that contains 15%DMSO (volume ratio) of 4 ℃ of following precoolings in 1: 2 (volume ratio) ratio, and the TS-2 composition is: Tris-Cl 10mM, pH 8.0, sucrose 110mM, KHCO
3100mM, hyclone 15% (volume ratio).
(3) seminal fluid-dilution mixed liquor need not 4 ℃ of balances, be sub-packed in 2 milliliters of frozen pipes (every pipe 1.5ml mixed liquor) after, directly freezing.
(4) undertaken freezingly by three-step approach, promptly earlier frozen pipe is placed liquid nitrogen surface top 6 centimeters balance 10min, balance 5min on liquid nitrogen surface drops in the liquid nitrogen at last and preserves then.
(5) the turbot sperm is preserved to thaw with check sperm freezing preservation effect after 1 day-1 year or be used for insemination in liquid nitrogen and is tested, when thawing, frozen pipe is balance 5 minutes in liquid nitrogen steam at first, follow quick-thawing in 37 ℃ of water-baths, sperm in frozen pipe all melts, during the needed time be 1.5-2min.Microscopy is found, is frozen smart motility rate all more than 70%.
(6) be 1000: 1 at smart ovum than respectively, 2000: 1 and 5000: 1 o'clock, do respectively and freeze smart and bright smart insemination experiment, under the situation of various smart ovum ratios, freeze smart rate of fertilization and the bright smart significant difference that also do not have with check.Found that smart ovum ratio is at 1000: 1 o'clock, freezing smart rate of fertilization is 47.9% ± 0.35, and what aquatic foods were smart is 56.4% ± 2.33, does not have significant difference.When smart ovum ratio is 5000: 1, freezing smart rate of fertilization is 52.1% ± 0.84, bright smart be 58.9% ± 16.0, do not have significant difference yet.
For confirming that frozen turbot sperm can be applied to actual production, we have done 7 a large amount of insemination experiments altogether, and (used turbot ovum amount is between 100-440ml, freeze essence (mixed liquor of seminal fluid-dilution) amount between 1.5-8ml, the result shows, freezing smart and bright essence does not all have significant difference on rate of fertilization and incubation rate, the fry of hatching has all reached the requirement of measuring in the actual production.Wherein once to illustrate, freeze essence (1.5ml) and the insemination of 240ml turbot ovum below with 1 pipe.Freeze smart rate of fertilization and incubation rate and be respectively 76.5% and 55.5%, and bright smart control group is not have significant difference between 76.5% and 44.2%, two group.
2. embodiment is preserved in the perch freezing of semen
(1) the perch seminal fluid also is to gather with the method that gently is pressed into ripe milter belly, and quality evaluating method is the same, and survival rate is higher than 80% perch sperm and is used for freezing and the insemination experiment.
(2) with the perch seminal fluid collected by 1: 1 (volume ratio) dilution proportion in the dilution that contains 20%DMSO (volume ratio) of 4 ℃ of precoolings, dilution prescription name is called MPRS, consists of: NaCl60.35mM, NaH
2PO
41.80mM, NaHCO
33.00mM, KCl 5.23mM CaCl
22H
2O1.13mM, MgCl
2.6H
2O 1.13mM, the mixed liquor of D-Glucose 55.55mM. perch seminal fluid-dilution are used for freezing preservation experiment behind 4 ℃ of following balance 30min.
(3) the perch seminal fluid-dilution mixed liquor behind the balance 30min is sub-packed in behind 2 milliliters of frozen pipes (every pipe 1.5ml) freezing immediately, when freezing, earlier frozen pipe is placed liquid nitrogen surface top 6 centimeters balance 10min, balance 5min on liquid nitrogen surface drops in the liquid nitrogen at last and preserves then.
(4) the perch sperm is preserved in liquid nitrogen after 1 day-1 year and is thawed, and earlier frozen pipe is proposed to place liquid nitrogen steam balance 5 minutes from liquid nitrogen when thawing, then quick-thawing in 37 ℃ of water-baths.Repeatedly microscopy found that, freezes smart survival rate between 70-80%.
(5) perch is frozen smart the preservation after three days in liquid nitrogen, the insemination of thawing, and when smart ovum ratio is 20000: 1,40000: 1,80000: 1 and 320000: 1 o'clock, what freeze polyspermism rate and incubation rate and aquatic foods essence did not all have a significant difference.The perch sperm is preserved 1 year in liquid nitrogen after, the insemination of thawing, under the situation of various smart ovum ratios, freezing the polyspermism rate does not have a significant difference with bright smart yet.Illustrate that freezing essence is can preserve in the liquid nitrogen midium or long term fully.
(6) perch is frozen smart a large amount of insemination experimental result.Being used in the 3.5ml perch that preserved a year in the liquid nitrogen freezes smart and the insemination of 230ml perch ovum, mid-term is calculated rate of fertilization at primitive gut in insemination back, freeze smart fertilization rate reached 84%, incubation rate reaches 90%, with the no significant difference of the smart control group of aquatic foods (96.8% ± 2.3 and 87.2% ± 3.1).
Two, save as example with freshwater fish grass carp seminal fluid superfreeze, embodiments of the present invention are described
(1) gamete is collected: contain the phase in the grass carp artificial propagation, push the belly of ripe milter, collect the grass carp seminal fluid with suction pipe.The grass carp ovum also is to obtain by the mode that gently is pressed into ripe raun belly.
(2) semen dilution: after in dilution, adding 20%DMSO, be placed on 4 ℃ of following precoolings, again with the seminal fluid gathered and said mixture dilution proportion with 1: 1.Used dilution is D-15, and its composition is: NaCl 136.75mM, KCl 6.71mM and glucose 83.33mM.Seminal fluid after the dilution is freezing behind the balance 20min in 4 ℃ of refrigerators.
(3) sperm freezing and thawing: after the mixed liquor of seminal fluid-dilution is sub-packed in the frozen pipe of 2ml (every pipe 1.5ml), at first put the liquid nitrogen surface top 6cm balance 10min of place, follow balance 5min on liquid nitrogen surface, drop in the liquid nitrogen at last.The grass carp sperm is preserved in liquid nitrogen after 2 days-2 years and is thawed.When thawing earlier with frozen pipe balance 5min in liquid nitrogen steam, quick-thawing in 37 ℃ of water-baths then.
(4) preserve 2 days grass carp sperm insemination result in liquid nitrogen: be used in the 1ml grass carp that preserved 2 days in the liquid nitrogen and freeze smart and the insemination of 15ml ovum, freezing smart group rate of fertilization and incubation rate is respectively 90.2% ± 4.7,90% ± 10, and control group is 98% ± 3.2 and 91% ± 4.5.Both there was no significant differences.
(5) preserve the grass carp sperm insemination result in 2 years in liquid nitrogen: be used in the 1ml grass carp that preserved 2 years in the liquid nitrogen and freeze smart and the insemination of 15ml ovum, freezing smart group rate of fertilization is 73.% ± 3.1, bright smart group be 88.5% ± 0.6, both differences are not remarkable.
The present invention and prior art contrast are characterized in: (1) dilution prescription is formed comparatively simple, and reagent source is convenient; (2) set up three steps cooling frozen mode, freezing preservation effect is stable, reliable, simple to operation, is easy to promote; (3) single tube spermatozoa cryopreservation amount is increased to the frozen pipe of 1.8ml from original 0.25ml straw, is suitable for setting up the sperm freezing storehouse and applies in fish production, genetic breeding and germplasm are preserved; (4) technology of the present invention's foundation all obtains stable result in more than the 10 kind of fish of being tested, and freezes smart motility rate and is stabilized in more than 65%, and rate of fertilization and incubation rate all reach bright smart level.
Claims (1)
1. the practical method preserved of a fish sperm superfreeze, the technology operation method that it is characterized in that it is, comprises thawing and inseminating of packing that fish sperm collection, sperm freezing preserve preparation, the dilution of selecting antifreeze, sperm for use and the balance of dilution, sperm and freezing, frozen semen:
Semen collection: towel off and do ripe milter gonopore zone, gently press belly, collect the seminal fluid of extruding with suction pipe.Place clean bottle.Seminal fluid is put short-term preservation on ice, behind the microscopy survival rate is used for preserving at the seminal fluid more than 80%.
Sperm freezing is preserved the preparation of dilution: fresh water cyprinid fish (comprising black carp, grass carp, silver carp, flathead, carp, crucian carp, bream etc.) semen diluent is D-15, and its prescription is: NaCl 135-136.75mM, KCl 6-6.71mM and glucose 83-83.33mM; Seawater fish turbot spermatozoa diluent is TS-2, and its prescription is: Tris-Cl 9-11mM, pH8.0, sucrose 105-115mM, KHCO
395-105mM, 9-11% hyclone (volume ratio); Perch, lefteye flounder and stone flounder spermatozoa diluent are MPRS, and its prescription is: NaCl 59.35-61.35mM, NaH
2PO
41.70-1.90mM, NaHCO
32.5-3.5mM, KCl 4.73-5.73mM, CaCl
22H
2O 0.63-1.63mM, MgCl
26H
2O 0.63-1.63mM, D-Glucose 50.55-60.55mM.
The antifreeze of selecting for use is DMSO, and its suitable concentration is 8-10%;
The dilution of sperm and balance: the dilution of black carp, grass carp, silver carp, flathead, carp, crucian carp, bream, perch, lefteye flounder and stone flounder seminal fluid and precooling in 4 ℃ of refrigerators carries out freezing behind 4 ℃ of refrigerator balance 20-30min after mixing in the ratio of 1: 1 or 1: 2 (volume ratio).Directly freezing after the turbot seminal fluid then dilutes, need not balance period.
The packing of sperm and freezing: the seminal fluid that balance is good, dilution 1.0-1.8ml are sub-packed in the frozen pipe of 2ml, carry out freezing preservation according to three step cooling patterns.I.e. 6 centimeters balance 10min above liquid nitrogen surface, balance 5min on liquid nitrogen surface drops in the liquid nitrogen then and preserves.Best rate of temperature fall is 31 ℃/min (16 ℃ to-15 ℃) and 18.6 ℃/min (from-12 ℃ to-180 ℃).
Thawing and insemination of frozen semen: when thawing, earlier frozen pipe is proposed to place liquid nitrogen steam balance 5 minutes from liquid nitrogen, from liquid nitrogen container, take out then, be placed on quick-thawing in 37 ℃ of waters.Freeze and carry out artificial insemination with bright ovum after essence is thawed, way is: frozen semen is fallen on ovum, and stirring and evenly mixing adds a little fresh water (cyprinid fish sperm) or seawater (seawater fish sperm) and activates sperm, makes it and ovum fertilization.Place incubator to hatch embryonated egg after the insemination.
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