CN102077823A - 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 59
- 238000000034 method Methods 0.000 title claims abstract description 40
- 230000009027 insemination Effects 0.000 title claims abstract description 22
- 238000005138 cryopreservation Methods 0.000 title claims abstract description 8
- 241000318729 Siniperca scherzeri Species 0.000 title abstract 5
- 238000007710 freezing Methods 0.000 claims abstract description 16
- 230000008014 freezing Effects 0.000 claims abstract description 16
- 238000010790 dilution Methods 0.000 claims abstract description 12
- 239000012895 dilution Substances 0.000 claims abstract description 12
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 239000003085 diluting agent Substances 0.000 claims abstract description 4
- 241000404975 Synchiropus splendidus Species 0.000 claims description 39
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 38
- 102000002322 Egg Proteins Human genes 0.000 claims description 23
- 108010000912 Egg Proteins Proteins 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 23
- 210000004681 ovum Anatomy 0.000 claims description 23
- 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
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 230000004720 fertilization Effects 0.000 claims description 13
- 238000004321 preservation Methods 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000010257 thawing Methods 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 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
- 239000008103 glucose Substances 0.000 claims description 3
- 230000004899 motility Effects 0.000 claims description 3
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- 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
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- 238000009395 breeding Methods 0.000 abstract description 4
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- 238000002474 experimental method Methods 0.000 abstract description 2
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- 241000264847 Siniperca chuatsi Species 0.000 abstract 2
- 241000264848 Siniperca Species 0.000 abstract 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 breed, particularly large-scale cultivation is bred.
Background technology
Current, be operated in various places at the genetic improvement of mandarin fish and extensively carry out.The mandarin fish resource fish of China distributes extensive relatively, but stock number is gradually reducing, and it is serious that bio-diversity is lost problem; Because the popularity that distributes, what the time difference of gonad maturity was big can reach two months, and breeding period is about 20 days, and hybridization 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, stirred and leave standstill 2-3 minute; 2. incubating oosperm process, the hatching in early stage: fertilized egg with pouring in the incubation plate after 4～8 ‰ the physiological saline sterilization, adds water purification and hatches in the hatching house, 16～20 ℃ of hatching house's temperature, wind sheltering, daylighting, avoid the sunlight direct projection, and change water and control water temperature; Final-period management: after fertilized egg beginning emerges, stop to change water and sterilization, add water purification, after 48 to 72 hours, the fry that begins flat trip is thrown in into breed pond.This invented technology adopts rationally, scientific methods, 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 one by one, 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 vigor can decline to a great extent as time passes; 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 big, and how utilizing spot mandarin fish seminal fluid efficiently is the problem of a key.In general reproductive process, often do not note smart ovum ratio, perhaps can not adjust smart ovum ratio flexibly 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 a kind of being useful for and solve the spot mandarin fish and stick up in the crossbreeding of mouth mandarin fish gonad development asynchronous, when guaranteeing fertilization rate, improve the method for spot mandarin fish sperm availability.
The present invention is achieved in that a kind of spot mandarin fish seminal fluid cryopreservation method, comprises the 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 the above-mentioned dilution balance and dilution with behind 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 placed liquid nitrogen liquid level top 5 centimeters balances 10 minutes, dropped into liquid nitrogen after 5 minutes in 1 centimeters balance above the liquid nitrogen surface again and preserved standby.
This method further comprises: carry out quality examination after the described semen collection step: the perusal normal semen is a milky, uses simple microscope, under 10 * 40 times, activates with clear water, and is standby greater than 70% seminal fluid preservation to average motility rate, otherwise, abandon it.
The present invention also comprises the test-tube method of a kind of spot mandarin fish, it is characterized in that comprising the steps:
(1) semen thawing: the frozen semen of claim 1 or 2 described method preparations is thawed, immerse after being about to that above-mentioned seminal fluid is slowly mentioned and leaving standstill 6 minutes from liquid nitrogen near liquid nitrogen container mouth liquid nitrogen steam to rock in 40 ℃ the warm water again and thaw 60 seconds, 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 back seminal fluid that thaws in right amount and activate fertilization with 0.4% NaCl solution.
This method further comprises: under 26 ℃ of room temperature environments, preserved ten minutes behind the described semen thawing, and artificial insemination again, promptly smart ovum fully mixes the back and activates fertilization with 0.4% NaCl solution.
The smart ovum quantitative proportion of described artificial insemination is that 1000000: 1 o'clock effects and cost are better.
A kind of spot mandarin fish semen freezing of the present invention is preserved and test-tube method has 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 freezing preservation and test-tube method hybridization 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, promptly 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 being easy to realize, can be used for general fish reproduction place; Can realize storage, solve spot mandarin fish and the nonsynchronous problem of mandarin fish gonad development in the hybridization spot mandarin fish resource; In reproductive process, can carry out the flexible adjustment of smart ovum ratio according to breeding demand and parent population stock number.
Description of drawings
Below in conjunction with accompanying drawing the semen freezing of a kind of spot mandarin fish of the present invention is preserved and test-tube method, embodiment is elaborated.Described embodiment is that the mode with non-limiting example provides:
Fig. 1: different smart ovum are than bright essence under the condition and freeze smart fertilization rate contrast
Embodiment
Embodiment one
About 2 milliliters of seminal fluid with freezing preservation two tail spot mandarin fish are example below, and specific operation process is described:
(1) semen collection: choose good spot mandarin fish parent population two tails of maturity; Towel off dried fish body belly; Push belly gently, wipe and wipe clean gonopore with dry towel or toilet paper after extruding a small amount of seminal fluid; Pushing belly more gently extrudes the seminal fluid of no urine and fecal pollution and with two 2 milliliters of centrifuge tubes collections, numbers B-1 and B-2 respectively.The seminal fluid of gathering is put on the ice face and temporarily preserves.
(2) semen quality inspection: the perusal normal semen is a milky; Drip clear water on the 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 the cover plate immediately, observe down at 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 by weighing 0.85 gram NaCl, 0.05 gram KCl respectively, 1.5 gram glucose are dissolved in the water, and add water and are settled to 100 milliliters, regulating pH value is 6.5, and it is standby to put into 4 ℃ of refrigerators after preparing.
(4) sperm dilution and packing: shake up behind the DMSO respectively at 0.8 milliliter of dilution of packing and 0.1 milliliter of 4 ℃ of preservation in 10 2 milliliters the frozen pipe, also blow and beat evenly in 0.2 milliliter of frozen pipe of adding of extracting extract respectively again, two milliliters are sub-packed in 10 frozen pipes, are put on the ice face lucifuge balance then 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, present embodiment is an antifreeze.
(5) semen freezing: the 5 centimeters balances 10 minutes above the liquid nitrogen liquid level of liquid nitrogen container of the frozen pipe after the balance, liquid nitrogen surface top 1 centimeters balance drops into the liquid nitrogen preservation after 5 minutes.
Embodiment two
Being used for insemination with the pipe freezing preservation seminal fluid that thaws below is example, and specific operation process is described:
(1) semen thawing: when thawing with slowly mention in the frozen Guan Congcong liquid nitrogen and near liquid nitrogen container mouth liquid 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 on ovum and add 1 milliliter of essence of freezing of having thawed 10 minutes, mix and stir 1 minute after, adds 10 milliliter of 0.4% Nacl solution and activates and be fertilized; Add water inflation hatching, primitive gut is added up fertilization rate and can reach 40% mid-term.
Embodiment three
With 0.5 milliliter of freezing preservation seminal fluid, be example than being used for insemination under the condition below, specific operation process be described at low smart ovum:
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 on ovum, adds 0.5 milliliter of essence of freezing of having thawed 10 minutes, mix and stir 1 minute after, adds 50 milliliter of 0.4% Nacl solution activation and is fertilized; Add water inflation hatching, can hatch fry 3000 tails.
Referring to Figure of description 1, different smart ovum are than bright smart under the condition and freeze smart fertilization rate contrast, and during artificial insemination, with freezing smart insemination, smart ovum ratio is the fertilization rate of essence ovum than 200000: 1 in the time of can obtaining approximate bright smart insemination at 1000000: 1 o'clock.When the smart ovum of aquatic foods compares greater than 200000: 1, freeze smart ovum ratio greater than 1000000: 1 o'clock, fertilization rate has only raising by a small margin.
Certainly, the invention is not restricted to address the embodiment shown in the accompanying drawing, modification and equivalent transformation that the spirit of Fan Yiben creation is done, or adopt various deformation on this basis, all belong in the protection domain of the present invention.
Claims (7)
1. spot mandarin fish seminal fluid cryopreservation method, its feature may further comprise the 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%, and regulating the pH value is 6.5;
(3) sperm dilution: with above-mentioned seminal fluid and dilution with behind 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 the above-mentioned dilution balance placed liquid nitrogen liquid level top 5 centimeters balances 10 minutes, dropped into liquid nitrogen after 5 minutes in 1 centimeters balance above the liquid nitrogen surface then and preserved standby.
2. spot mandarin fish seminal fluid cryopreservation method according to claim 1 is characterized in that:
Carry out quality examination after the described semen collection step: the perusal normal semen is a milky, uses simple microscope, under 10 * 40 times, activates with clear water, and is standby greater than 70% seminal fluid preservation to average motility rate, otherwise, abandon it.
3. the test-tube method of spot mandarin fish is characterized in that comprising the steps:
(1) semen thawing: the frozen semen of claim 1 or 2 described method preparations is thawed, immerse again after being about to that above-mentioned seminal fluid is slowly mentioned and near liquid nitrogen container mouth liquid nitrogen steam, leaving standstill 6 minutes from liquid nitrogen to rock in 40 ℃ the warm water and thaw 60 seconds, the seminal fluid after thawing be put in keep in Dark Place under the room temperature stand-by;
(2) artificial insemination: choose the female parent population that is in the ecbolic effect phase, manually squeeze ovum in clean anhydrous container, adds the above-mentioned seminal fluid that thaws in right amount after, activate with 0.4% NaCl solution and to be fertilized.
4. the test-tube method of spot mandarin fish according to claim 3 is characterized in that: under 26 ℃ of room temperature environments, preserved ten minutes behind the described semen thawing, and artificial insemination again, promptly smart ovum fully mixes the back and activates fertilization with 0.4%NaCl solution.
5. according to claim 3 or the test-tube method of 4 described spot mandarin fishes, it is characterized in that: the smart ovum quantitative proportion of described artificial insemination is 1000000: 1.
6. according to claim 3 or the test-tube method of 4 described spot mandarin fishes, it is characterized in that: described female parent population is for sticking up the mouth mandarin fish.
7. the test-tube method of spot mandarin fish according to claim 6 is characterized in that: the smart ovum quantitative proportion of described artificial insemination is 1000000: 1.
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Cited By (13)
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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 |
| CN104396943A (en) * | 2014-11-28 | 2015-03-11 | 陈小洁 | Pangasias sutchi semen storage method |
| 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 |
| 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 |
| CN110036950B (en) * | 2019-04-23 | 2021-07-30 | 青岛农业大学 | Artificial crossbreeding method for mariculture of rainbow trout |
| CN113875748A (en) * | 2021-11-18 | 2022-01-04 | 武汉市农业科学院 | In-vitro preservation solution for mandarin fish eggs and application thereof |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104396943A (en) * | 2014-11-28 | 2015-03-11 | 陈小洁 | Pangasias sutchi semen storage method |
| 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 |
| CN110036950B (en) * | 2019-04-23 | 2021-07-30 | 青岛农业大学 | Artificial crossbreeding method for mariculture of rainbow trout |
| 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 |
| CN113875748A (en) * | 2021-11-18 | 2022-01-04 | 武汉市农业科学院 | In-vitro preservation solution for mandarin fish eggs and application thereof |
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