CN103548730A - Method for inducing and collecting high-quality sperms of haliotis discus hannai - Google Patents
Method for inducing and collecting high-quality sperms of haliotis discus hannai Download PDFInfo
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- CN103548730A CN103548730A CN201310517084.1A CN201310517084A CN103548730A CN 103548730 A CN103548730 A CN 103548730A CN 201310517084 A CN201310517084 A CN 201310517084A CN 103548730 A CN103548730 A CN 103548730A
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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
Abstract
The invention belongs to the technical field of marine organisms and particularly relates to a method for inducing and collecting high-quality sperms of haliotis discus hannai. The method comprises the steps of selection of high-quality male parent abalones, drying stimulation of the parent abalones in shade, preparation of ozone seawater, sperm collection, sperm motility detection and determination of the fertilization rate and the hatchability. According to the method, 3-4-year plump male parent abalones with gonads mature are selected, drying stimulation in shade is performed, the parent abalones are placed in seawater under the ultraviolet irradiation and arranged in the quiet and dark environment so as to release the sperms, and the high-concentration sperms are collected by centrifugation. The collected abalone sperms are subjected to artificial insemination, and the fertilization rate is over 95%. Practice proves that the abalone sperms collected in the abalone collection mode can be applied to production time and research. The method for inducing and collecting the high-quality sperms of the haliotis discus hannai is of great significance for artificial breeding, germplasm conservation and genetic diversity protection and the like of the haliotis discus hannai.
Description
Technical field
The invention belongs to marine biotechnology field, specifically the sperm induction of a kind of wrinkle Pan Abalone essence high-quality and collecting method.
Background technology
Haliotis discus hannai Ino (Haliotis discushannai) belongs to Gastropoda, Archaeogastropoda ，Bao section.The large-scale original gastropod in ocean of algae feeding habits, one of principal item of northern China mariculture shellfish, its individuality is larger.Bao meat is fertile, is the treasure in marine products, supplies luxurious hotel and outlet more.Except eating raw, can be processed into can or dried abalone, fetch long price.Awabi shell is the famous Chinese medicine shell of seaear, is again the important materials of making shell picture, and market is larger to the demand of wrinkle Pan Abalone.In recent years, due to the continuous expansion of cultivation scale, the continuous expansion of wrinkle Pan Abalone artificial breeding in addition, and the structure etc. of extensive family in scientific research, though aborning or in research work to the demand of wrinkle Pan Abalone high-quality seminal fluid also constantly expansion.A little less than the sperm viability obtaining due to wrinkle Pan Abalone dissection, cannot carry out artificial insemination, wrinkle Pan Abalone discharges seminal fluid naturally in addition, and the time is fixing, and wayward, the cultivation that has seriously restricted wrinkle Pan Abalone is bred.The artificial induction of wrinkle Pan Abalone high-quality sperm and the foundation of collecting method can be induced at any time and to impel its ejaculation at male haliotis discus hannai Ino breeding period; and little to close Bao injury; can repeatedly utilize; overcome close unfixed shortcoming of Bao ejaculation time, be conducive to the protection of hybridization, seed selection and the gene diversity of wrinkle Pan Abalone.The aquatic foods essence that simultaneously obtains high-quality high concentration is to set up the prerequisite of its sperm super-low temperature freezing store method, and induction and the collecting method of setting up haliotis discus hannai Ino high-quality sperm are that Haliotisdiscushannaisperm sperm refrigeration preservation lays the foundation.Therefore, set up wrinkle Pan Abalone semen collection method significant to the protection of the development of mariculture industry and bio-diversity.
Summary of the invention
The object of the present invention is to provide the sperm induction of a kind of wrinkle Pan Abalone essence high-quality and collecting method.
For achieving the above object, the technical scheme that the present invention takes is:
The sperm induction of a kind of wrinkle Pan Abalone essence high-quality and collecting method:
A. the selection of high-quality parent Bao, the sexual gland of selecting natural waters to propagate artificially is full and be milky white, faint yellow male close Bao or the male close Bao of being propagated artificially by natural waters is full and be milky white, faint yellow male close Bao through the gland of nourishing one's nature temporarily;
B. close Bao dry in the shade stimulation is pulled male close Bao out from storage pond, holding pond, and the outside of belly is lucifuge dry in the shade stimulation 1.5-2 hour upward;
C. the male close Bao after above-mentioned dry in the shade stimulation is placed in through ultraviolet ray and irradiated and be warming up to the seawater of 20-21 ℃, under dark surrounds, stimulate 0.5-1 hour, until ejaculation;
D. by the centrifugal collection turbid solution of the sperm of above-mentioned collection, obtain haliotis discus hannai Ino sperm.
Described step is a) in 5-6 month, selects upset that natural waters is propagated artificially to observe that sexual gland is full, swelling, presents male close Bao in milky white, flaxen 3-4 age; Or the 3-4 age of selecting natural waters to propagate artificially, select the male close Bao of the high 12-15cm of shell at 18-19 ℃, the density of supporting temporarily with every cubic metre of water body 20-25 is supported temporarily, support temporarily 3-7 days, treat that male close Bao sexual gland is full, swelling, present milky white, faint yellow and male Bao and ingest, move normal.
Describedly change water every day while supporting temporarily, and throw something and feed sea-tangle and/undaria pinnitafida, average every male Bao 10-15g/ days.
Described step b) male close Bao is pulled out from storage pond, holding pond, the outside of belly is placed upward, infiltrates and is screwed onto half-dried towel, fixing, lucifuge dry in the shade stimulation 1.5-2 hour.
Describedly fresh seawater is exposed to seawater under the uviol lamp of 15W be warming up to 20-21 ℃, then the male close Bao of the 1.5-2h that dries in the shade is placed in one.
Further, get under the uviol lamp of 50L fresh seawater with 15W and irradiate 2.5h, heating rod rises to 20-21 ℃ by water temperature by 18-19 ℃ simultaneously, then the male close Bao of the 1.5-2h that dries in the shade is placed in one.
Described steps d is processed 8-10 minute by collecting sperm with the centrifugal speed of 3000-3500r/min, abandons supernatant, collects bottom turbid solution, is the seminal fluid of high concentration, and sperm concentration: 106-107, is stored in 0-4 ℃.
The above-mentioned seminal fluid of collecting is passed through to computer-assisted analysis (CASAS) rate of motion higher than 95%, averaged curve movement velocity is higher than 90.0 μ m/s, and sperm curvilinear velocity is all higher than 50 μ m/s, and more than 25% sperm curvilinear velocity is higher than 100 μ m/s.
Described haliotis discus hannai Ino seminal fluid is 80-100 with the number ratio of the ovum of the female close Bao of induction: 1, and mix in the fresh filtering sea that is placed on 20 ℃ and mix, after 2h, get fertilized egg, detect fertilization rate; The 10-15 of the volume that wherein amount of fresh filtering sea is ovum doubly.
Tool of the present invention has the following advantages:
1. the present invention selects the well-developed bull parent of sexual gland Bao, by simple induction, can obtain good wrinkle dish Abalone sperm;
2. little to close Bao injury by induction mode of the present invention, close Bao can reuse, and has reduced cost;
3. high-quality sperm of the present invention is induced required instrument, equipment, simple, facilitate feasible, easy operating;
4. the present invention induces acquisition mode not to be subject to site limitation, at wrinkle Pan Abalone, between sexual maturing period, all can induce ejaculation whenever and wherever possible, and the sperm viability that induction is discharged is high, convenient and swift;
5. by the method, can obtain the haliotis discus hannai Ino sperm of high concentration, high-quality, conveniently carry out the structure of extensive artificial insemination, family;
6. the sperm quality of fertilization is good, and test-tube fertilization rate, incubation rate are high;
Embodiment
Embodiment 1:
May is on the occasion of wrinkle dish Abalone reproduction period, obtains the end that 3-4 propagates artificially age broadcast wrinkle dish Abalone with the precious plant in the Lao East Sea, Huang Island Jiangnan, selects the high 12-15cm of shell, and sexual gland is full, swelling, presents flaxen male close Bao Jinhang and supports temporarily; Support temporarily temperature 18-19 ℃, support temporarily 20, every cubic metre of water body of density, throw something and feed sea-tangle and/or undaria pinnitafida, average every male Bao 10-15g/ days, changes water every day once.And support temporarily 3-7 days, to male Bao, ingest, move and be normal, sexual gland milky, faint yellow and full.Male close Bao is pulled out from storage pond, holding pond, and the outside of belly is put in the plastics hollow out basket of 30cm * 20cm upward, by investing above male close Bao with infiltrating and being screwed onto half-dried towel, is fixed to and can not at will rocks.Then be positioned over the quiet region of lucifuge, dry in the shade stimulation 1.5 hours.
And the 50L fresh seawater of preprepared being irradiated under the uviol lamp of 15W to 2.5h, heat up 2 ℃ simultaneously, water temperature is in 18-19 ℃ of seawater that is upgraded to 20-21 ℃.
Then the male close Bao of the 1.5h that dries in the shade is placed in to the seawater that ultraviolet ray is irradiated and heated up, under peace and quiet, dark surrounds, induces 0.5 hour, male abalone is ejaculation successively.
The seminal fluid of the seawater the inside of collection discharge centrifugal 10 minutes with 3000r/min, collects the muddy seminal fluid in bottom.The seminal fluid of collecting is 2.6 * 107 through computer-assisted analysis (CASAS) density, rate of motion 99%%%, averaged curve movement velocity is higher than 97.4.0 μ m/s, and sperm curvilinear velocity is all higher than 50 μ m/s, and 32% sperm curvilinear velocity is higher than 100 μ m/s.The ovum of the sperm of the male close Bao that induction is discharged and the female close Bao of induction is by smart ovum 80-100: 1 number, than mixing, is mixed in 20 ℃ of fresh filtering seas, and the amount of seawater is approximately 10-15 times of above-mentioned ovum volume.After 2h, get at random more than 100 ovum at every turn, get three times, statistics fertilization rate, 14h adds up incubation rate, gets at random each more than 100 of larvas, gets three times.The quantity that wherein fertilization rate refers to start the ovum of division accounts for the percentage of the quantity of ovum altogether; Incubation rate refers to hatch the percentage that accounts for total fertilized egg to embryo's quantity of trochophore.The above-mentioned abalone sperm fertilization rate reached to 98.5% of collecting, 97.8%, 97.0%, average 97.8%; Incubation rate 95.4%, 90.5%, 94.0%, average out to 93.2%.
Embodiment 2:
June is obtained the high 9-12cm of shell at Jiangnan aquatic farm, sexual gland is full, swelling, present flaxen male close Bao, from culturing pool, pull 15 male Baos out, the outside of belly is put in the white plastic foam box of 50cm * 20cm upward, by being screwed onto half-dried towel with the infiltration of ultraviolet seawater, invest above male close Bao, make it at will to rock.Be positioned over the quiet region of lucifuge, dry in the shade stimulation 1.5 hours, getting power is the 50L fresh seawater of 15W ultra violet lamp 3h, the male close Bao of dry in the shade stimulation 1.5h is placed in to ultraviolet ray irradiated and heated up 2 ℃, be in the seawater of 20-21 ℃, be put in peace and quiet, dark surrounds stimulation 0.5 hour, observe close Bao Luxu ejaculation.Collection seminal fluid centrifugal 10 minutes with 3000r/min, collects the muddy seminal fluid in bottom.The seminal fluid of collecting is through computer-assisted analysis (CASAS), density is 5.6 * 106, rate of motion 100%%%, and averaged curve movement velocity is higher than 98.7 μ m/s, and sperm curvilinear velocity is all higher than 50 μ m/s, and 43%% sperm curvilinear velocity is higher than 100 μ m/s.The ovum of the sperm of the male close Bao that induction is discharged and the female close Bao of induction is in smart ovum number 80-100: 1 ratio is mixed (sperm: ovum), be added to 20 ℃ of fresh filtering seas after mixing, the amount of seawater is approximately 10-15 times of amount of ovum.After 2h, get at random more than 100 ovum at every turn, get three times, statistics fertilization rate, 14h adds up incubation rate, gets at random each more than 100 of larvas, gets three times.The quantity that wherein fertilization rate refers to start the ovum of division accounts for the percentage of the quantity of ovum altogether; Incubation rate refers to hatch the percentage that accounts for total fertilized egg to embryo's quantity of trochophore.The above-mentioned abalone sperm fertilization rate reached to 97.4% of collecting, 96.4%, 95.1%, average 96.3%; Incubation rate 92.4%, 90.3%, 92.7%, average out to 91.8%.
Claims (7)
1. wrinkle dish Abalone essence high-quality sperm is induced and a collecting method, it is characterized in that:
A. the selection of high-quality parent Bao, the sexual gland of selecting natural waters to propagate artificially is full and be milky white, faint yellow male close Bao or the male close Bao of being propagated artificially by natural waters is full and be milky white, faint yellow male close Bao through the gland of nourishing one's nature temporarily;
B. close Bao dry in the shade stimulation is pulled male close Bao out from storage pond, holding pond, and the outside of belly is lucifuge dry in the shade stimulation 1.5-2 hour upward;
C. the male close Bao after above-mentioned dry in the shade stimulation is placed in through ultraviolet ray and irradiated and be warming up to the seawater of 20-21 ℃, under dark surrounds, stimulate 0.5-1 hour, until ejaculation;
D. by the centrifugal collection turbid solution of the sperm of above-mentioned collection, obtain haliotis discus hannai Ino sperm.
2. by wrinkle Pan Abalone essence high-quality claimed in claim 1 sperm induction and collecting method, it is characterized in that: described step is a) in 5-6 month, select upset that natural waters is propagated artificially to observe that sexual gland is full, swelling, present male close Bao in milky white, flaxen 3-4 age;
Or the 3-4 age of selecting natural waters to propagate artificially, select the male close Bao of the high 12-15cm of shell at 18-19 ℃, the density of supporting temporarily with every cubic metre of water body 20-25 is supported temporarily, supports temporarily, swelling full to sexual gland, presents male close Bao in milky white, flaxen 3-4 age.
3. by the sperm induction of wrinkle Pan Abalone essence high-quality and collecting method described in claim 1 or 2, it is characterized in that: describedly change water every day while supporting temporarily, and throw something and feed sea-tangle and/undaria pinnitafida, average every male Bao 10-15g/ days.
4. by wrinkle Pan Abalone essence high-quality claimed in claim 1 sperm induction and collecting method, it is characterized in that: described step b) male close Bao is pulled out from storage pond, holding pond, the outside of belly is placed upward, infiltrates and is screwed onto half-dried towel, fixing, lucifuge dry in the shade stimulation 1.5-2 hour.
5. by wrinkle Pan Abalone essence high-quality claimed in claim 4 sperm induction and collecting method, it is characterized in that: described in
Fresh seawater is exposed to seawater under the uviol lamp of 15W and be warming up to 20-21 ℃, then the male close Bao of the 1.5-2h that dries in the shade is placed in one.
6. by wrinkle Pan Abalone essence high-quality claimed in claim 1 sperm induction and collecting method, it is characterized in that: described steps d is processed 8-10 minute by collecting sperm with the centrifugal speed of 3000-3500r/min, abandons supernatant, collects the muddy sperm in bottom, is stored in 0-4 ℃.
7. by wrinkle Pan Abalone essence high-quality claimed in claim 1 sperm induction and collecting method, it is characterized in that: described haliotis discus hannai Ino seminal fluid is 80-100 with the number ratio of the ovum of the female close Bao of induction: 1, mix in the fresh filtering sea that is placed on 20 ℃ and mix, after 2h, get fertilized egg, detect fertilization rate; The 10-15 of the volume that wherein amount of fresh filtering sea is ovum doubly.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104430087A (en) * | 2014-11-27 | 2015-03-25 | 中国水产科学研究院黑龙江水产研究所 | Method for obtaining high-quality Hucho taimen sperms through screening |
| CN111387103A (en) * | 2020-03-25 | 2020-07-10 | 大连海宝渔业有限公司 | Method for intensively producing sperm from hybrid first-generation male abalone of Haliotis diversicolor |
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Cited By (2)
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| CN104430087A (en) * | 2014-11-27 | 2015-03-25 | 中国水产科学研究院黑龙江水产研究所 | Method for obtaining high-quality Hucho taimen sperms through screening |
| CN111387103A (en) * | 2020-03-25 | 2020-07-10 | 大连海宝渔业有限公司 | Method for intensively producing sperm from hybrid first-generation male abalone of Haliotis diversicolor |
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