CN102258006B - Method for storing spermatophore of litopenaeus vannamei at low temperature - Google Patents
Method for storing spermatophore of litopenaeus vannamei at low temperature Download PDFInfo
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- CN102258006B CN102258006B CN 201110129658 CN201110129658A CN102258006B CN 102258006 B CN102258006 B CN 102258006B CN 201110129658 CN201110129658 CN 201110129658 CN 201110129658 A CN201110129658 A CN 201110129658A CN 102258006 B CN102258006 B CN 102258006B
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Abstract
The invention discloses a method for storing spermatophore of litopenaeus vannamei at low temperature. The method comprises the following steps of: acquiring the spermatophore, fixing the spermatophore, storing the spermatophore and dyeing spermatozoa. In the method, a culture layer for the spermatophore of the litopenaeus vannamei is prepared from 0.3 to 0.4 part of 1.8 to 3.0 percent self-prepared type I liquid rat tail collagen serving as a medium for contracting the culture layer, 0.3 to 0.4 part of culture solution of 2*1,640, 0.1 to 0.2 part of fetal calf serum in an American GIBCO company and 0.1 part of Matrigel (a soluble basilar membrane matrix) in an American Sigma company, wherein the culture layer is viscous at normal temperature, and can be solidified at the temperature of 40 DEG C and can wrap the spermatophore. The spermatophore is packaged in separate an epoxy resin (EP) tube at the normal temperature, the spermatophore is wrapped by the prepared culture layer, and the EP tube wrapped with the spermatophore is arranged in a refrigerator to be stored at the low temperature of 4 DEG C. The method has the characteristics of simplicity of operation, complete storage of a spermatophore structure, high survival rate of the spermatozoa, long retention time, stable effect of low-temperature storage and the like and is easy to popularize and apply. The retention time of the spermatophore is at least 3 months, and the survival rate of the spermatozoa is over 80 percent.
Description
Technical field
The present invention relates to method, particularly a kind of Environment of Litopenaeus vannamei Low spermatophore low temperature store method that a kind of animal sperm low temperature is preserved.
Background technology
Environment of Litopenaeus vannamei Low (Litopenaeus vannamei) has another name called Penaeus Vannmei, is one of world scale cultured prawn kind, is the highest prawn of China's output.Though more and more important on aquaculture, its sperm is preserved technology still seldom, limited the feasibility of selecting breeding greatly.At present, the method that the sperm freezing of mammal and fish is preserved adopts the nitrogen ultra low temperature freezing more, sperm freezing preservation technology exists freezes that smart motility rate is not high enough, fertilization rate is not high, it is little to freeze smart reserve capacity, do not reach problems such as practicability level, and because the sperm of the sperm of prawn and other animals is different, the spermatium of prawn is provided the spermatophore parcel of its nutriment.Because the existence of spermatophore, spermatophore can form ice crystal when liquid nitrogen cryogenics was preserved, and caused the death of sperm.Abroad, Chow etc. (1985) are balance Macrobrachium rosenbergii 15-30min in running water and 10% glycerol mixture, and the teat glass of then spermatophore being transferred to 1mL is frozen in liquid nitrogen.This method has just confirmed can play the protective effect of part to spermatophore by the glycerine balanced action, but can not stop spermatophore to form the ice crystal process.It is freezeproof protectant that Dumont etc. (1992) find with dimethyl sulfoxide (DMSO) (DMSO), in liquid nitrogen with 1.6 ℃ of min
-1The freezing Penaeus Vannmei sperm of speed than 0.3 ℃ of min
-1With 1.8 ℃ of min
-1Better effect is arranged.Ke Yafu etc. (1996) can avoid prawn spermatogenesis acrosome reaction with the freezing Chinese prawn sperm of dilution that contains 10%DMSO and 5～10% glycerine, discovery Chinese prawn sperm at 0-4 ℃ of balance 20～30min.Though this method has been preserved sperm, in handling the spermatophore process, sperm viability is exerted a certain influence, and the sperm that removes spermatophore is difficult to carry out fertilization process after recovery, survival rate only reaches 60%.Therefore this method can not satisfy the condition that the sperm that utilizes preservation carries out follow-up study work fully.
Summary of the invention
The objective of the invention is: preserve for the preservation of Environment of Litopenaeus vannamei Low germplasm, genetic diversity protection, genetic breeding and production provide a kind of simple to operate, spermatophore structure that complete, sperm survival rate height, holding time are long, the Environment of Litopenaeus vannamei Low spermatophore low temperature store method of practicability.
The present invention realizes by following technical scheme:
The method that Environment of Litopenaeus vannamei Low spermatophore low temperature is preserved, comprise that spermatophore collection, spermatophore are fixed, spermatophore is preserved and the staining of sperm process, method is: under normal temperature condition spermatophore is sub-packed in EP and manages, adopt the culture layer parcel spermatophore of preparation, to wrap up spermatophore EP pipe and place refrigerator, preserve under 4 ℃ low temperature, specific operation process is:
(1) spermatophore collection: left hand is held the prawn afterbody on the superclean bench in aseptic iuntercellular, and the right hand is held head, separates last to step with right hand forefinger then, and left hand thumb extruding spermatophore is put into the EP pipe earlier with the spermatophore of gathering.
(2) spermatophore is fixed: on the superclean bench under the normal temperature condition in aseptic iuntercellular each spermatophore is sub-packed in the import EP pipe, adds preparation culture layer parcel spermatophore fast, seal with sealing film then.
(3) spermatophore is preserved: the EP pipe of (2) step parcel spermatophore is positioned in 4 ℃ of interior antistaling boxes of cell culture chamber.
(4) staining of sperm and be calculated to be motility rate: extrude seminal fluid gently and by dilution in 1: 10, the sperm suspension of getting 0.9mL moves in the test tube, 0.4% the trypan blue solution that adds 0.1mL, mixing, after 3 minutes it is splashed in the blood cell counting plate, examine under a microscope painted sperm number and sperm sum.Sperm survival rate=(sperm sum-blue number of sperm)/sperm sum * 100%.
Above-described spermatophore is preserved, and per 1 week takes out 1 spermatophore and observes its survival rate with trypan blue dyeing, puts into the volume number of culture layer along with the size of spermatophore is regulated, and guarantees that the sperm of preserving more than 3 months survives.
Above-described culture layer, have fixing and provide spermatophore required nutriment, its formula components, volume parts and compound method are: 0.3～0.4 part of concentration is the liquid mouse tail collagen of self-control I type and 0.3～0.4 part of 2 * RPMI-1640 of 1.8～3.0%, 0.1 the hyclone of～0.2 part of U.S. GIBCO company, 0.1 the Matrigel (a kind of solubility basement membrane matrix) of part U.S. Sigma company, be loaded in the EP pipe that is placed on ice chest, abundant mixing, the NaOH solution of adding 0.1% is regulated pH value 7.2～7.4.
Above-described culture fluid: 1 bag of 1640 medium of employing U.S. GIBCO company, the salinity that fully is dissolved in the 500ml filtration sterilization is in 30 ‰ the seawater, be mixed with 2 * RPMI-1640 that concentration is 20.8g/L, and add 2.38g HEPES (HEPES) buffer in solution to its final concentration be 10mmol/L, add with aseptic 1mol/L NaOH and regulate between pH to 6.8～7.4, it is 1,000,000 units that adding penicillin and streptomycin are regulated its final concentration, filter filtration through 0.22 μ m is distributed into the 50mL/ bottle again, and is standby.
The liquid mouse tail of above-described self-control I type collagen, with rat mouse tail, place 75% alcohol to soak 30min, the rat tail skin of under aseptic condition, tearing, tail is cut into several sections, extract white tail tendon out, immerse in 0.1% the acetic acid of 150ml, place 4 ℃ of refrigerators, and stir with magnetic stirring apparatus, behind the 48h with the centrifugal collection supernatant of the speed of 3000r/min both collagen solution, calculate the concentration of prepared collagen solution, concrete grammar is: get a filter paper and fully dry back title filter paper weight in baking oven, be designated as W0; The liquid collagen of preparation is dripped several in filter paper, weigh, be designated as W1; Fully dry in baking oven and weigh dripping the filter paper that liquid collagen is arranged, be designated as W2, the concentration of the prepared collagen that can be asked by following formula:
Collagen concentration=(W2-W0)/(W1-W0)
Advantage of the present invention and good effect:
That the present invention has is simple to operate, the spermatophore structure is preserved the characteristics complete, that the sperm survival rate is high.The prawn spermatophore is kept in 4 ℃ the aseptic culture layer, this culture layer has fixing and the effect of nutrition is provided.The result shows that 1.8～3.0% mouse tail collagen is suitable for the medium that shrinks as culture layer, and the holding time of spermatophore was at least more than 3 months, and sperm survival rate reaches more than 80%.
Embodiment
Below by embodiment, to the further specific description of technical scheme of the present invention.
Embodiment 1:
One, fix and provide the preparation of the required nutriment culture layer of spermatophore:
(1) culture fluid:
With 1 bag of 1640 medium of U.S. GIBCO company, the salinity that fully is dissolved in the 500ml filtration sterilization is in 30 ‰ the seawater, be mixed with 2 * RPMI-1640 that concentration is 20.8g/L, and add 2.38g HEPES (HEPES) buffer in solution to its final concentration be 10mmol/L, add with aseptic 1mol/L NaOH and regulate pH 7.2, it is 1,000,000 units that adding penicillin and streptomycin are regulated its final concentration, filter filtration through 0.22 μ m is distributed into the 50mL/ bottle again, and is standby.
(2) the liquid mouse tail of self-control I type collagen:
Get 1 on rat mouse tail, place 75% alcohol to soak 30min, the rat tail skin of tearing under aseptic condition is cut into several sections with tail, extract white tail tendon out, immerse in 0.1% the acetic acid of 150ml, place 4 ℃ of refrigerators, and stir with magnetic stirring apparatus, both got collagen solution with the centrifugal collection supernatant of the speed of 3000r/min behind the 48h, calculate the concentration of prepared collagen solution, concrete grammar is: get a filter paper and fully dry back title filter paper weight in baking oven, be designated as W0; The liquid collagen of preparation is dripped several in filter paper, weigh, be designated as W1; Fully dry in baking oven and weigh dripping the filter paper that liquid collagen is arranged, be designated as W2, the concentration of collagen, standby.
(3) culture layer:
The liquid mouse tail collagen of drawing 0.4mL 1.8% with measuring pipette respectively with 2 * 1640 concentrates of 0.4mL, 0.1mL hyclone (U.S. GIBCO company), the Matrigel of 0.1mL U.S. sigma company (a kind of solubility basement membrane matrix) of U.S. Sigma company is loaded in the EP pipe of the 1.5mL that is placed on ice chest, fully mixing adds 0.1%NaOH solution and regulates pH value 7.2.
Two, low temperature is preserved operating process
Left hand is held the prawn afterbody on the superclean bench under the normal temperature condition in aseptic iuntercellular, and the right hand is held head, separates last to step with right hand forefinger then, and left hand thumb extruding spermatophore is put into the EP pipe earlier with the spermatophore of gathering.On the superclean bench under the normal temperature condition in aseptic iuntercellular each spermatophore is sub-packed in the import EP pipe, adds preparation culture layer parcel spermatophore fast, seal with sealing film then.The EP pipe of parcel spermatophore is positioned in 4 ℃ of antistaling boxes in the cell culture chamber.The spermatophore of preserving, per 1 week takes out 1 spermatophore and observes its survival rate with trypan blue dyeing, puts into the volume number of culture layer along with the size of spermatophore is regulated, and detects the sperm survival rate of preserving and reaches 80.6% in 4 months.
Embodiment 2:
One, fix and provide the preparation of the required nutriment culture layer of spermatophore:
(1) culture fluid:
With 1 bag of 1640 medium of U.S. GIBCO company, the salinity that fully is dissolved in the 500ml filtration sterilization is in 30 ‰ the seawater, be mixed with 2 * RPMI-1640 that concentration is 20.8g/L, and add 2.38g HEPES (HEPES) buffer in solution to its final concentration be 10mmol/L, add with aseptic 1mol/L NaOH and regulate pH7.3, it is 1,000,000 units that adding penicillin and streptomycin are regulated its final concentration, and the filter filtration through 0.22 μ m is distributed into the 50mL/ bottle again, and is standby.
(2) the liquid mouse tail of self-control I type collagen:
Get 1 on rat mouse tail, place 75% alcohol to soak 30min, the rat tail skin of tearing under aseptic condition is cut into several sections with tail, extract white tail tendon out, immerse in 0.1% the acetic acid of 150ml, place 4 ℃ of refrigerators, and stir with magnetic stirring apparatus, both got collagen solution with the centrifugal collection supernatant of the speed of 3000r/min behind the 48h, calculate the concentration of prepared collagen solution, concrete grammar is: get a filter paper and fully dry back title filter paper weight in baking oven, be designated as W0; The liquid collagen of preparation is dripped several in filter paper, weigh, be designated as W1; Fully dry in baking oven and weigh dripping the filter paper that liquid collagen is arranged, be designated as W2, the concentration of collagen.
(3) culture layer:
Draw the liquid mouse tail collagen of 0.4mL 3.0% and 2 * RPMI-1640 of 0.3mL with measuring pipette, 0.2mL the hyclone of U.S. GIBCO company, 0.1mL the Matrigel of U.S. Sigma company is loaded in the EP pipe of the 1.5mL that is placed on ice chest, fully mixing adds and drips 0.1%NaOH solution adjusting pH value 7.3.
Two, low temperature is preserved operating process
Left hand is held the prawn afterbody on the superclean bench under the normal temperature condition in aseptic iuntercellular, and the right hand is held head, separates last to step with right hand forefinger then, and left hand thumb extruding spermatophore is put into the EP pipe earlier with the spermatophore of gathering.On the superclean bench under the normal temperature condition in aseptic iuntercellular each spermatophore is sub-packed in the import EP pipe, adds preparation culture layer parcel spermatophore fast, seal with sealing film then.The EP pipe of parcel spermatophore is positioned in 4 ℃ of antistaling boxes in the cell culture chamber.The trypan blue exclusion method is identified the sperm that survives.The spermatophore of preserving, per 1 week takes out 1 spermatophore and observes its survival rate with trypan blue dyeing, puts into the volume number of culture layer along with the size of spermatophore is regulated, and detects the sperm survival rate of preserving and reaches 80.6% in 4 months.
Claims (2)
1. the Environment of Litopenaeus vannamei Low spermatophore low temperature method of preserving, comprise that spermatophore collection, spermatophore are fixed, spermatophore is preserved and the staining of sperm process, it is characterized in that: under normal temperature condition, spermatophore is sub-packed in EP and manages, adopt the culture layer parcel spermatophore of preparation, to wrap up spermatophore EP pipe and place refrigerator, preserve under 4 ℃ low temperature, specific operation process is:
(1) spermatophore collection: left hand is held the prawn afterbody on the superclean bench in aseptic iuntercellular, and the right hand is held head, separates last to step with right hand forefinger then, and left hand thumb extruding spermatophore is put into the EP pipe earlier with the spermatophore of gathering;
(2) spermatophore is fixed: on the superclean bench under the normal temperature condition in aseptic iuntercellular each spermatophore is sub-packed in the EP pipe, adds preparation culture layer parcel spermatophore fast, seal with sealing film then;
(3) spermatophore is preserved: the EP pipe of (2) step parcel spermatophore is positioned in 4 ℃ of interior antistaling boxes of cell culture chamber;
(4) staining of sperm: extrude seminal fluid gently and dilute by 1:10, the sperm suspension of getting 0.9mL moves in the test tube, 0.4% the trypan blue solution that adds 0.1mL, mixing, after 3 minutes it is splashed in the blood cell counting plate, examine under a microscope painted sperm number and sperm sum, calculate the sperm survival rate;
Described culture layer has fixing and provides spermatophore required nutriment, its formula components, volume parts and compound method are: 0.3～0.4 part of concentration is the liquid mouse tail collagen of self-control I type and 0.3～0.4 part of 2 * RPMI-1640 that concentration is 20.8g/L of 1.8～3.0%, 0.1 the hyclone of～0.2 part of U.S. GIBCO company, 0.1 the Matrigel of part U.S. Sigma company is loaded in the EP pipe that is placed on ice chest, abundant mixing, the NaOH solution of adding 0.1%, regulate pH value 7.2～7.4, standby;
Described culture fluid adopts 1 bag of 1640 medium of U.S. GIBCO company, the salinity that fully is dissolved in the 500ml filtration sterilization is in 30 ‰ the seawater, be mixed with 2 * RPMI-1640 that concentration is 20.8g/L, and add the 2.38gHEPES buffer in solution to its final concentration be 10mmol/L, add with aseptic 1mol/LNaOH and regulate between pH to 6.8～7.4, it is 1,000,000 units that adding penicillin and streptomycin are regulated its final concentration, filter filtration through 0.22 μ m is distributed into the 50mL/ bottle again, and is standby;
The liquid mouse tail of described self-control I type collagen is with rat mouse tail, place 75% alcohol to soak 30min, the rat tail skin of under aseptic condition, tearing, tail is cut into several sections, extracts white tail tendon out, immerse in 0.1% the acetic acid of 150ml, place 4 ℃ of refrigerators, and stir with magnetic stirring apparatus, behind the 48h with the centrifugal collection supernatant of the speed of 3000r/min both collagen solution, calculate the concentration of prepared collagen solution.
2. store method according to claim 1, it is characterized in that: described spermatophore is preserved, per 1 week takes out 1 spermatophore and observes its survival rate with trypan blue dyeing, puts into the volume number of culture layer along with the size of spermatophore is regulated, and guarantees that the sperm of preserving more than 3 months survives.
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| CN103232969B (en) * | 2013-05-06 | 2014-07-09 | 中国海洋大学 | Prawn cell culture medium |
| CN104996328B (en) * | 2015-06-18 | 2017-12-19 | 广东海洋大学 | A kind of Penaeus Vannmei comes off the artificial implantation method again of spermatophore |
| CN113455497B (en) * | 2021-07-21 | 2022-04-12 | 山东省海洋科学研究院 | Ultralow temperature cryopreservation method for sperm of penaeus chinensis |
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Non-Patent Citations (8)
| Title |
|---|
| Chilled storage of white shrimp (Litopenaeus vannamei) spermatophores;Subuntith Nimrat 等;《Aquaculture》;20061201;第261卷(第3期);第945页第4段至第946页第3段 * |
| Cryopreservation of aquatic invertebrate semen: a review;J-C Gwo;《Aquaculture Research》;20000331;第31卷(第3期);第259页–271页 * |
| J-C Gwo.Cryopreservation of aquatic invertebrate semen: a review.《Aquaculture Research》.2000,第31卷(第3期), |
| Subuntith Nimrat 等.Chilled storage of white shrimp (Litopenaeus vannamei) spermatophores.《Aquaculture》.2006,第261卷(第3期), |
| 中国对虾精子超低温保存的研究;柯亚夫 等;《海洋与湖沼》;19960331;第27卷(第2期);第187页至第193页 * |
| 对虾精荚和精子的研究进展;赵连翠 等;《海洋科学》;20050209;第29卷(第2期);第73页至第77页 * |
| 柯亚夫 等.中国对虾精子超低温保存的研究.《海洋与湖沼》.1996,第27卷(第2期), |
| 赵连翠 等.对虾精荚和精子的研究进展.《海洋科学》.2005,第29卷(第2期), |
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