CN102077823B - Method for cryopreservation of semen of siniperca scherzeri and artificial insemination - Google Patents
Method for cryopreservation of semen of siniperca scherzeri and artificial insemination Download PDFInfo
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- 210000000582 semen Anatomy 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 36
- 230000009027 insemination Effects 0.000 title claims abstract description 20
- 238000005138 cryopreservation Methods 0.000 title abstract description 15
- 241000318729 Siniperca scherzeri Species 0.000 title abstract 5
- 238000010790 dilution Methods 0.000 claims abstract description 12
- 239000012895 dilution Substances 0.000 claims abstract description 12
- 238000007710 freezing Methods 0.000 claims abstract description 12
- 230000008014 freezing Effects 0.000 claims abstract description 12
- 239000003085 diluting agent Substances 0.000 claims abstract description 4
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 38
- 241000404975 Synchiropus splendidus Species 0.000 claims description 34
- 102000002322 Egg Proteins Human genes 0.000 claims description 21
- 108010000912 Egg Proteins Proteins 0.000 claims description 21
- 210000004681 ovum Anatomy 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 20
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 230000004720 fertilization Effects 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 239000000243 solution Substances 0.000 claims description 8
- 238000010257 thawing Methods 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 230000002528 anti-freeze Effects 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
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- 230000004899 motility Effects 0.000 claims description 3
- 239000011435 rock Substances 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 238000007865 diluting Methods 0.000 claims description 2
- 230000002196 ecbolic effect Effects 0.000 claims description 2
- 230000001105 regulatory effect Effects 0.000 claims description 2
- 241000251468 Actinopterygii Species 0.000 abstract description 15
- 230000008569 process Effects 0.000 abstract description 6
- 238000009402 cross-breeding Methods 0.000 abstract description 5
- 210000002149 gonad Anatomy 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 241000264847 Siniperca chuatsi Species 0.000 abstract 2
- 241000264848 Siniperca Species 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 230000012447 hatching Effects 0.000 description 9
- 238000004321 preservation Methods 0.000 description 6
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- 238000009396 hybridization Methods 0.000 description 2
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- 238000009400 out breeding Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 230000032696 parturition Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 206010022998 Irritability Diseases 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
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Abstract
The invention belongs to the technical field of fish reproduction and relates to a method for ultralow-temperature cryopreservation of semen of siniperca scherzeri and artificial insemination in crossbreeding, which can be widely applied in culture of siniperca chuatsi in families and groups, especially large-scale culture and reproduction. The method comprises the steps of semen collection, diluent preparation, semen dilution, semen freezing, semen unfreezing, artificial insemination and the like. In the artificial insemination, as the optimum ratio of frozen sperms to fresh spawn is determined to be 1000000:1 by different experiments about sperm-spawn ratio. In the invention, the requirement for equipment is low, the operation is easy to realize, the method can be used for common fish reproduction fields, can realize storage of siniperca scherzeri resources, solves the problem of asynchronous growth of gonads of the siniperca chuatsi and the siniperca scherzeri in crossbreeding of siniperca, and increases the utilization ratio of the sperms of the siniperca scherzeri, and in the reproduction process, the sperm-spawn ratio can be flexibly adjusted according to the breeding requirements, the production need and the amount of parent fishes.
Description
Affiliated technical field
The present invention relates to the fish reproduction technology; belong to technical field of aquaculture, be specifically related to the cryopreservation method of spot mandarin fish seminal fluid, and with the test-tube method of freezing spot mandarin fish seminal fluid; can be widely used in family, collective's mandarin fish cultivation, particularly large-scale cultivation is bred.
Background technology
Current, be operated in various places for the genetic improvement of mandarin fish and extensively carry out.The mandarin fish resource fish of China distributes relatively extensive, but stock number is gradually reducing, and bio-diversity loss problem is serious; Due to the popularity that distributes, what the time difference of gonad maturity was large can reach two months, and breeding period is about 20 days, and crossing work is higher to the requirement of maturity of fish gonads, overdone or not full maturity all will have a strong impact on crossbreeding breeding fertilization rate.
Be the technical problem of artificial insemination and hatching in the solution fish reproduction, people have carried out broad research and experiment.
Patentee: Yang Xin, patent (the patent No.: CN200810018840.5) of the title " silverfish artificial insemination and oosperm hatching technique " of application on January 28th, 2008, open (bulletin) day: 2008.07.16, open (bulletin) number: CN101218899), this invention relates to a kind of fish reproduction technical field, especially a kind of silverfish artificial insemination and oosperm hatching technique, have 1. artificial insemination processes: seminal fluid and ovum grain are clamp-oned container, add 4～8 ‰ physiological saline, stir and standing 2-3 minute; 2. incubating oosperm process, the hatching in early stage: fertilized egg with pouring in incubation plate after 4～8 ‰ physiological saline sterilization, adds water purification and hatches in the hatching house, 16～20 ℃ of hatching house's temperature, wind sheltering, daylighting, avoid direct sunlight, and change water and control water temperature; Final-period management: after fertilized egg beginning emerges, stop changing water and sterilization, add water purification, after 48 to 72 hours, the fry that begins flat trip is thrown in into culturing pool.This invented technology adopts rationally, the method for science, has increased substantially fry output and quality.
The suchlike innovation and creation that relate to Technique in Fishes insemination and oosperm hatching technique, correlation technique also has a lot, does not just enumerate, and in fish culture propagation technique field, these technical schemes success beyond doubt are feasible,
The cryopreservation method of fish sperm is mature on the whole, but species variation obviously exists; The time that spot mandarin fish seminal fluid is preserved at ambient temperature is very short, and along with the passage of time vigor can decline to a great extent; With respect to mandarin fish, spot mandarin fish stock number is few, and small, 1 milliliter of the common less than of the sperm volume that each tail fish can provide, and also the irritability in reproductive process is large, and how utilizing efficiently spot mandarin fish seminal fluid is the problem of a key.In general reproductive process, often do not note smart ovum ratio, perhaps can not adjust flexibly smart ovum ratio according to the amount of sperm volume or ovum, reduce the unnecessary wasting of resources in the time of with the raising fertilization rate.
Summary of the invention
The purpose of this invention is to provide and a kind ofly be practically applicable to solve the spot mandarin fish and stick up in the crossbreeding of mouth mandarin fish gonad development asynchronous, improve the method for spot mandarin fish sperm availability when guaranteeing fertilization rate.
The present invention is achieved in that a kind of spot mandarin fish seminal fluid cryopreservation method, comprises the following steps:
(1) semen collection: gather the good spot mandarin fish parent population seminal fluid of maturity, deposit under the environment of 4 ℃ of cleanings standby;
(2) diluent preparing: preparation contains the solution of NaCl 0.85% (weight ratio), KCl 0.05%, glucose 1.5%, regulates the pH value and is 6.5, and is standby;
(3) sperm dilution: with the seminal fluid after above-mentioned dilution balance and dilution with after 1: 4 dilution ratio mixed diluting, add antifreeze DMSO to final concentration be 9%, and be put under 4 ℃ of conditions balance 25 minutes;
(4) semen freezing: above-mentioned dilution seminal fluid was placed in liquid nitrogen liquid level top 5 centimeters balance 10 minutes, then dropped into liquid nitrogen after 5 minutes in 1 centimeters balance above liquid nitrogen surface and save backup.
The method further comprises: carry out quality examination after described semen collection step: visually observing normal semen is milky, uses simple microscope, under 10 * 40 times, activates with clear water, average motility rate is saved backup greater than 70% seminal fluid, otherwise, abandon it.
The present invention also comprises a kind of test-tube method of spot mandarin fish, it is characterized in that comprising the following steps:
(1) semen thawing: the frozen semen of the described method preparation of claim 1 or 2 is thawed, be about to that above-mentioned seminal fluid is slowly mentioned and immerse after standing 6 minutes to rock in the warm water of 40 ℃ near liquid nitrogen container oral fluid nitrogen steam again thaw 60 seconds from liquid nitrogen, the seminal fluid after thawing is put in room temperature and keeps in Dark Place stand-by;
(2) artificial insemination: choose the female parent population of ecbolic effect phase, manually squeeze ovum in clean anhydrous container, add the above-mentioned rear seminal fluid that thaws in right amount and activate fertilization with 0.4% NaCl solution.
The method further comprises: preserved ten minutes under 26 ℃ of room temperature environments after described semen thawing, then artificial insemination, namely smart ovum fully mixes rear use 0.4% NaCl solution activation fertilization.
Described artificial insemination essence ovum quantitative proportion is that 1000000: 1 o'clock effects and cost are better.
A kind of spot mandarin fish semen cryopreservation of the present invention and test-tube method have following beneficial effect:
1. employing seminal fluid cryopreservation method, the time difference the Nature condition that solves inhomogeneity mandarin fish gonad maturity can't obtain the hybridization of inhomogeneity mandarin fish and get excellent problem, promotes kind diversification and optimum organization, and suitable large-scale cultivation is bred.
2. spot mandarin fish semen cryopreservation and test-tube method crossing work overcome the requirement of maturity of fish gonads higher, overdone or not full maturity all will have a strong impact on the problem of outbreeding fertilization rate, improve the outbreeding fertilization rate, namely solve mandarin fish and the nonsynchronous problem of spot mandarin fish gonad development in the mandarin fish crossbreeding and improve the method for spot mandarin fish sperm availability.
3. the present device requirement is low, and operation is easy to realize, can be used for general fish reproduction place; Can realize the storage to spot mandarin fish resource, solve spot mandarin fish and the nonsynchronous problem of mandarin fish gonad development in hybridization; Can carry out the flexible adjustment of smart ovum ratio according to breeding demand and parent population stock number in reproductive process.
Description of drawings
To the present invention's a kind of spot mandarin fish semen cryopreservation and test-tube method, embodiment is elaborated below in conjunction with accompanying drawing.Described embodiment is that the mode with non-limiting example provides:
Fig. 1: different smart ovum are than bright essence under condition and freeze smart fertilization rate contrast
Embodiment
Embodiment one
The below as example, illustrates specific operation process take approximately 2 milliliters of seminal fluid of freezing preservation two tail spot mandarin fish:
(1) semen collection: choose good spot mandarin fish parent population two tails of maturity; Towel off dried fish body belly; Push gently belly, wipe and wipe clean gonopore with dry towel or toilet paper after extruding a small amount of seminal fluid; Push gently again belly and extrude without the seminal fluid of urine and fecal pollution and collect with two 2 milliliters of centrifuge tubes, number respectively B-1 and B-2.The seminal fluid that gathers is put on the ice face and temporarily preserves.
(2) semen quality inspection: visually observing normal semen is milky; Drip clear water on slide, and be ready to cover glass, a little immerses in the preprepared clear water and activates with the toothpick picking, use simple microscope after cover plate immediately, observe under 10 * 40 times, repeat twice, average motility rate is greater than 70% the follow-up freezing preservation that is used for.
(3) diluent preparing: take respectively 0.85 gram NaCl, 0.05 gram KCl, 1.5 gram glucose are dissolved in the water, add water and be settled to 100 milliliters, regulating pH value is 6.5, puts into 4 ℃ of refrigerators after preparing standby.
(4) sperm dilution and packing: shake up after the DMSO respectively at 0.8 milliliter of dilution of packing and 0.1 milliliter of 4 ℃ of preservation in the cryopreservation tube of 10 2 milliliters, again respectively 0.2 milliliter of extracting extract add in cryopreservation tube and piping and druming evenly, two milliliters are sub-packed in 10 cryopreservation tubes, then are put on the ice face lucifuge balance 25 minutes.Antifreeze DMSO is dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide or DMSO), is that molecular formula is (CH
3)
2The chemical substance of SO, it is a colourless liquid, the present embodiment is antifreeze.
(5) semen freezing: the 5 centimeters balance 10 minutes above the liquid nitrogen liquid level of liquid nitrogen container of the cryopreservation tube after balance, liquid nitrogen surface top 1 centimeters balance drops into the liquid nitrogen preservation after 5 minutes.
Embodiment two
The below is used for insemination as example take the pipe freezing preservation seminal fluid that thaws, and specific operation process is described:
(1) semen thawing: when thawing with a cryopreservation tube from slowly mention from liquid nitrogen and near liquid nitrogen container oral fluid nitrogen steam buffering immerse again after 6 minutes to rock in the warm water of 40 ℃ of preprepareds and thaw 60 seconds.Seminal fluid after thawing is put in room temperature (26 ℃) and keeps in Dark Place stand-by.
(2) insemination: choose female parent population one tail that the aftereffect phase of hastening parturition has arrived, the artificial ovum that squeezes is in clean anhydrous bowl, get 10 milliliters, ovum and add 1 milliliter of essence of freezing of having thawed 10 minutes, after mixing and stir 1 minute, add 10 milliliter of 0.4% Nacl solution and activate fertilization; Add water inflation hatching, primitive gut is added up fertilization rate and can reach 40% mid-term.
Embodiment three
The below is example at low smart ovum than being used for insemination under condition with 0.5 milliliter of freezing preservation seminal fluid, and specific operation process is described:
Choose female parent population one tail that the aftereffect phase of hastening parturition has arrived, manually squeeze ovum in clean anhydrous bowl, get 50 milliliters, ovum, adds 0.5 milliliter of essence of freezing of having thawed 10 minutes, after mixing and stir 1 minute, adds 50 milliliter of 0.4% Nacl solution activation and be fertilized; Add water inflation hatching, can hatch fry 3000 tails.
Referring to Figure of description 1, different smart ovum are than bright essence under condition and freeze the fertilization rate contrast of essence, and during artificial insemination, with freezing the essence insemination, smart ovum ratio is the fertilization rate of smart ovum than 200000: 1 in the time of can obtaining approximate bright essence insemination at 1000000: 1 o'clock.Than greater than 200000: 1, freeze smart ovum ratio greater than 1000000: 1 o'clock when the smart ovum of aquatic foods, fertilization rate only has raising by a small margin.
Certainly, the invention is not restricted to address the embodiment shown in accompanying drawing, modification and equivalent transformation that the spirit of Fan Yiben creation is done, or adopt on this basis various deformation, all belong in protection domain of the present invention.
Claims (4)
1. test-tube method of spot mandarin fish, its feature comprises the following steps:
(1) semen collection: gather the good spot mandarin fish parent population seminal fluid of maturity, deposit under the environment of 4 ℃ of cleanings standby;
(2) diluent preparing: preparation contains the solution of NaCl0.85%, KCl0.05%, glucose 1.5%, and above-mentioned percentage is g/100ml, and regulating the pH value is 6.5;
(3) sperm dilution: with above-mentioned seminal fluid and dilution with after 1: 4 dilution ratio mixed diluting, add antifreeze DMSO to final concentration be 9%, and be put under 4 ℃ of conditions balance 25 minutes;
(4) semen freezing: the seminal fluid after above-mentioned dilution balance was placed in liquid nitrogen liquid level top 5 centimeters balance 10 minutes, then dropped into liquid nitrogen after 5 minutes in 1 centimeters balance above liquid nitrogen surface and saved backup;
(5) semen thawing: frozen semen is thawed, be about to that above-mentioned seminal fluid is slowly mentioned and immerse again after standing 6 minutes to rock in the warm water of 40 ℃ near liquid nitrogen container oral fluid nitrogen steam thaw 60 seconds from liquid nitrogen, preserved ten minutes under 26 ℃ of room temperature environments after thawing;
(6) artificial insemination: choose the female parent population that is in the ecbolic effect phase, manually squeeze ovum in clean anhydrous container, after adding the above-mentioned seminal fluid that thaws in right amount, activate fertilization with 0.4%NaCl solution.
2. method according to claim 1 is characterized in that:
Carry out quality examination after described semen collection step: visually observing normal semen is milky, uses simple microscope, under 10 * 40 times, activates with clear water, average motility rate is saved backup greater than 70% seminal fluid, otherwise, abandon it.
3. method according to claim 1 is characterized in that: described artificial insemination essence ovum quantitative proportion is 1000000: 1.
4. according to claim 1,2 or 3 test-tube methods of described spot mandarin fish, it is characterized in that: described female parent population is for sticking up the mouth mandarin fish.
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| CN102258007A (en) * | 2011-09-02 | 2011-11-30 | 大连海洋大学 | Refrigerant and method for storing Pacific codfish sperms |
| CN102273439A (en) * | 2011-09-06 | 2011-12-14 | 中国水产科学研究院黄海水产研究所 | Cryopreservation and application method of Convict grouper sperms |
| CN102726369A (en) * | 2012-06-18 | 2012-10-17 | 中国水产科学研究院黄海水产研究所 | Ultralow temperature cryopreservation application method of Epinephelus oanceolutus sperms |
| CN104145865A (en) * | 2014-08-18 | 2014-11-19 | 池州市秋浦特种水产开发有限公司 | Breeding method of hybrid mandarin fishes |
| CN104396944A (en) * | 2014-11-28 | 2015-03-11 | 陈小洁 | Special diluent for pangasias sutchi semen |
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| CN104521943A (en) * | 2014-12-09 | 2015-04-22 | 南京师范大学 | Ultralow-temperature refrigeration preservation and recovery method for oxyeleotris marmorata semen |
| CN104814005A (en) * | 2015-03-26 | 2015-08-05 | 谢光玉 | Preservation method for seminal fluid of Schizothorax davidi |
| CN105265362A (en) * | 2015-11-16 | 2016-01-27 | 江西省水产科学研究所 | Cross breeding method for improving growth traits of slender mandarinfish |
| CN105360037A (en) * | 2015-12-23 | 2016-03-02 | 苏州市阳澄湖现代农业产业园特种水产养殖有限公司 | Siniperca scherzeri culturing method |
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| CN110495410A (en) * | 2019-08-15 | 2019-11-26 | 浙江省农业科学院 | A kind of little yellow croaker frozen sperm and Larimichthys crocea hybridizing propagation process |
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