CN109566490B - Method for cultivating triploid loaches - Google Patents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0604—Whole embryos; Culture medium therefor
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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 discloses a method for cultivating triploid loaches, which comprises the following steps: (a) fertilizing the loach sperms and the loach ova to form fertilized ova; (b) 2-4min after fertilization, treating the fertilized eggs for 1.5-5min, preferably 3min by using hydrostatic shock of 35-50 MPa; (c) and hatching the fertilized eggs to obtain the triploid loaches.
Description
Technical Field
The invention belongs to the field of aquatic science, and relates to a method for cultivating triploid loaches.
Background
Paramisgurnus dabryanus and Paramisgurnus dabryanus are respectively related to Cypriniformes, Cobitidae Paramisgurnus and Misgurnus, have similar shapes and often exist in the same domain, and are widely distributed in mainland China, Taiwan region China, and Semiaquillaena, Japan, Russia, India and the like. The Taiwan loach is one of the paramisgurnus dabryanus, and has a larger body, a cylindrical body and a shorter head compared with loaches. The Taiwan loach has delicious taste, rich nutrition, high protein content and low fat content. Compared with loaches, Taiwan loaches have the advantages of no mud drilling, fast growth, large specification, short culture period, easy domestication, high yield and the like.
Taiwan loaches are common small fishes in the family of the order of the Cyprinidae in China, frequently inhabit shallow water areas such as ditches, ditches and paddy fields with much silt, and are omnivorous benthic fishes; the fish can be properly grown at the water temperature of 20-30 ℃, can be breathed by gills, skins and intestines, has better resistance to hypoxia environment than other fishes, and is a breeding variety with larger production potential. The Taiwan loach has tender meat, delicious taste, rich nutrition and higher medicinal and dietetic therapy value. China exports loaches in large quantity every year, particularly, China has large demand in Japan and Korea, exports about 1 ten thousand every year, and has a value of 2000 ten thousand dollars.
Disclosure of Invention
In order to solve the defects of the prior art, the first aspect of the patent provides a cultivation method of triploid loaches, which comprises the following steps:
(a) fertilizing the loach sperms and the loach ova to form fertilized ova;
(b) 2-4min after fertilization, treating the fertilized eggs for 1.5-5min, preferably 3min by using hydrostatic shock of 35-50 MPa;
(c) and hatching the fertilized eggs to obtain the triploid loaches.
In some embodiments, the loach is selected from taiwan loach.
In some embodiments, the loach sperm is obtained by pressing the abdomen of a mature male loach.
In some embodiments, in the step (a), the loach ovum is obtained by:
(1) injecting 800-1200IU/kg bw of chorionic gonadotropin and 3-6 mu g/kg of luteinizing hormone releasing hormone analogue into the base part of the pectoral fin of the female loach;
(2) allowing the injected loaches to move in water for 6-12 hours;
(3) and extruding the belly of the loach to obtain the loach ovum.
In some embodiments, in step (a), the fertilization is a dry fertilization.
In some embodiments, (b) the fertilized egg of loach is treated 3min after fertilization with hydrostatic pressure of 35, 40, 45 or 50 MPa.
In some embodiments, the hydrostatic pressure operating step is: and mixing the activated loach fertilized eggs with water, placing the mixture in a pressure container, and applying hydrostatic pressure to the activated loach fertilized eggs and the water by applying mechanical pressure to the pressure container.
In a second aspect the invention provides the use of a breeding method according to the first aspect of the invention for accelerating the breeding pace of loaches.
The hydrostatic pressure method is adopted to induce the result to show that: 3min after insemination, hydrostatic pressure of 40MPa is adopted, shock treatment is carried out for 3min, and the triploid induction rate is the highest and reaches 35.6 percent. The DNA content and the chromosome number of the triploid fry cells are detected by adopting a flow cytometer and chromosome preparation, and the result shows that: the cell DNA content of the triploid fry is 1.5 times of that of the diploid control group fry; the chromosome number of the diploid fish fry is 2 n-48, and the chromosome number of the triploid fish fry is 3 n-72. Triploid Taiwan loach grows rapidly.
Drawings
FIG. 1 shows DNA content distribution diagrams of triploid (lower panel) and diploid (upper panel) Taiwan loach.
FIG. 2 is a chromosome analysis chart of diploid (a) and triploid (b) young Taiwan loach.
FIG. 3 is a photograph showing morphological characteristics of a triploid loach (lower panel) and a normal diploid individual (upper panel).
FIG. 4 is a photograph showing the ovarian development characteristics of a triploid loach (upper panel) and a normal diploid individual (lower panel).
Detailed Description
In order to better explain the technical scheme of the invention, the following detailed description of the embodiment of the invention is combined with the accompanying drawings. The following examples are intended to further illustrate the invention but should not be construed as being limitations or restrictive thereon. Unless otherwise specified, technical features used in the embodiments may be replaced with other technical features known in the art having equivalent or similar functions or effects without departing from the inventive concept.
The research adopts a hydrostatic pressure method to search and compare the difference of the induction effects of the two methods (the common heat shock method (processing at 39-42 ℃ for 2 minutes) is not suitable for the Taiwan loach, the survival rate is below 5 percent) and simultaneously adopts a flow cytometer to identify the cell DNA content of the triploid loach and the diploid loach and adopts a chromosome analysis technology to analyze the chromosome number of the triploid loach, thereby proving the feasibility of the technology, aiming at researching and perfecting the artificial induction method of the triploid Taiwan loach and providing basic data for the artificial induction and the actual production of the triploid of the Taiwan loach.
1 materials and methods
1.1 materials
The experimental fish is Taiwan loach (Taiwan loach is one of Paramisgurnus dabryanus, which is a shoal river mostly distributed in the middle and lower reaches of Yangtze river and in the northwest of Taiwan island in China, Paramisgurus dabryanus (Misgurnus anguillicaudatus) belongs to the cypriniformes, loach family and Misgurni genus), individuals with the weight of about 1000g, good body types, sexual maturity and good development are selected, the individuals are injected by adopting the basal part of a disposable pectoral fin, chorionic gonadotropin (HCG) and luteinizing hormone releasing hormone analogue (LHRH-A2) are mixed for induced spawning, the dosage is 1000IUHCG +5 mu g LHRH-A2/kg body weight, female fish injected with an induced spawning medicament is placed in a circular pool, the female fish is subjected to spunlace stimulation, the parent fish is pulled after 10 hours, and ova is obtained by adopting a method of manually extruding belly; the injection dosage of the male Taiwan loach is reduced by half (the dosage is 500IUHCG +2.5 mu g LHRH-A2/kg). The abdomen of the mature male fish was slowly squeezed and a clean white sperm was squeezed out.
1.2 Induction method
Inducing by hydrostatic pressure: the hydrostatic pressure is achieved by applying hydrostatic pressure to the aqueous solution of the fertilized egg to inhibit the second polar body of the fertilized egg from being discharged, rather than directly pressing the fertilized egg. The method comprises the steps of manually collecting mature ova and semen at the temperature of 23 +/-1 ℃, mixing the fertilized ova and aerated tap water in a pressure container after 3min after dry fertilization, plugging a piston into an opening of the pressure container, putting the pressure container into a press, pressurizing to corresponding pressure (40Mpa, 45Mpa, 50Mpa, 55Mpa and 60Mpa) within 15 seconds, releasing pressure to zero within 5 seconds after 3min treatment, inhibiting discharge of a second diode, doubling chromosomes of the ova, combining 5 test groups, enabling 2 groups to be parallel, respectively incubating in different incubation barrels, and cleaning a filter screen of the incubation barrel for 1 time every 1-2 h until seedlings emerge. The appropriate treatment pressure was determined by calculating the normal rate of emergence and the rate of triploid induction for each experimental combination.
And simultaneously setting a diploid control group (the male and the female of the Taiwan loach) (the control is artificial fertilization and hatching without hydrostatic pressure induction treatment).
1.4.1DNA content determination
The young Taiwan loach which is induced and treated by shock for 5 months is taken, a 1mL injector is soaked by a heparin sodium solution, the tail vein draws blood about 0.2mL,adding into EP tube containing 1mL LPBS, mixing, centrifuging at 1000rpm for 5min, discarding supernatant, washing and precipitating with PBS for 2 times, adding 1mL precooled (4 deg.C) PBS-ethanol stationary liquid, dispersing cells, standing in 4 deg.C refrigerator overnight for 18h, centrifuging at 1000rpm for 5min, discarding supernatant, adding 400 μ LPBS solution, mixing, adding 50 μ LPI staining solution and RNase solution, treating for 30min, and adopting BDAccuriTMDetection was performed by a flow cytometer type C6. And selecting chicken blood cells (the DNA content is 2.30pg) as a reference standard to carry out DNA content determination on the triploid of the Taiwan loach. The triploid inductivity is determined by the ratio of the triploid detected individual number to the total detected individual number.
1.4.2 chromosome preparation
And respectively fasting and temporarily culturing the triploid Taiwan loach and the normal diploid Taiwan loach bream detected by the ploidy analyzer in the aquarium for 3 days. Injecting PHA at the base of pectoral fin according to the dose of 10 mug/g (weight of fish), injecting colchicine according to the dose of 4 mug/g (weight of fish) after 22h, cutting off branchial rakes at two sides of the fish for bloodletting after 4h, taking head and kidney of the fish after 15min, putting the head and kidney into a small glass beaker containing physiological saline (0.75%) for the fish, washing off blood on the surface of the fish, and adding 4-6 mL of physiological saline for repeatedly tearing to fully release lymphocytes; filtering the suspension into a 6mL glass centrifuge tube by using a 300-mesh screen, centrifuging for 5min at 1000rpm, and removing supernatant; adding 6ml of 0.5% KCl solution (prepared in situ, preheated in a thermostat at 37 ℃), hypotonicizing at 37 ℃ for 50min, centrifuging at 1000rpm for 5min, and removing supernatant; adding 6mL of stationary liquid (methanol: glacial acetic acid ═ 3:1) (prepared in situ, precooled in a refrigerator at-20 ℃), centrifuging for 5min at 1000rpm after 20min, discarding the supernatant, and repeating the fixing and centrifuging for 2 times by the same method; and finally, sufficiently and uniformly mixing the rest 1-2 mL of fixing solution, dripping, drying in an oven at room temperature or 37 ℃ for 1h, dyeing by using Besojimsa dyeing solution, washing by using ultrapure water, and photographing and counting under a microscope after drying.
1.5 data analysis
The emergence rate and the triploid rate of the induced fries of each group are counted by SPSS software, One-Way ANOVA (One-Way ANOVA) is carried out, and multiple comparisons of data among the induced groups are carried out by using Least Significant Difference (LSD).
2 results
2.1 hydrostatic pressure Induction
As shown in table 1, the emergence rate of the fertilized eggs of the loaches in taiwan under hydrostatic pressure induction is significantly different (P <0.01) with different treatment pressures, and the emergence rate of the fries is decreased with the increase of the pressure. The influence of different pressures on the triploid induction rate of the Taiwan loaches can be known (table 1), and the difference of the influence of the shock treatment of 40Mpa and 45Mpa on the emergence rate of the Taiwan loaches is not obvious. The triploid induction rate difference of the two groups is obvious (P is less than 0.01), the triploid rate of the Taiwan loach induced by 40MPa of hydrostatic pressure is 85.6%, and the triploid rate of the Taiwan loach induced by 45MPa of hydrostatic pressure is 40.2% (Table 1). Comprehensively considering 2 factors of emergence rate and induction rate, the best effect is achieved when the Taiwan loach triploid is induced by hydrostatic pressure and shocked for 3min under 40MPa pressure after fertilization.
TABLE 1 hydrostatic pressure on the induction of Taiwan loach triploid
2.2 determination of the relative DNA content of the triploid fry of Taiwan loach
Through detection, the DNA content of the triploid Taiwan loach is 1.5 times of that of the normal diploid Taiwan loach parent.
30 loaches from the triploid Taiwan were sampled and subjected to DNA content detection using a CyFlow ploidy analyzer of Partec, Germany. The relative DNA content of triploid taiwan loaches was 50.15 ± 2.42(n ═ 30 tails) (fig. 1, supra), while the DNA content of normal diploid taiwan loach parents was 33.43 ± 1.15(n ═ 30 tails) (fig. 1, infra). Calculating with chicken erythrocyte standard to obtain triploid Taiwan loach blood cell DNA absolute content of 3.90 + -0.05 pg (M + -SD); the Taiwan loach of the normal individuals is 2.60 +/-0.02 pg (M +/-SD). The result shows that the DNA content of the triploid Taiwan loach is 1.5 times of that of the normal diploid Taiwan loach parent.
2.3 chromosome analysis of triploid fry of Taiwan loach
Normal diploid loach and triploid loach obtained by flow cytometry detection are respectively prepared into chromosomes, and the results are shown in fig. 2. From the results, the number of chromosomes of the diploid fry of the taiwan loach was 2n to 48 (as shown in (a)); the number of chromosomes of the triploid fish fry subjected to shock treatment is 3 n-72 (shown as (b)), and the result is consistent with the detection result of a ploidy analyzer; the karyotype analysis result shows that the karyotype formula of the normal diploid Taiwan loach is 2 n-24 m +20sm +4st, and NF-92; the nuclear type formula of the triploid Taiwan loach is 3 n-36 m +30sm +6st, and NF-138. The normal Taiwan loach has two sets of chromosome groups, and each set of chromosome group comprises 24 chromatids; and the triploid Taiwan loach has a three-set chromosome set, and has one more chromosome set compared with the normal diploid Taiwan loach.
2.4 growth characteristics of Taiwan loach triploid
The body length and the body weight of the 3-year-old adult Taiwan loaches are measured by sampling statistics, and the results show that the average body length of the triploid Taiwan loaches is 20.3 +/-5.6 cm, the body weight of the triploid Taiwan loaches is 88.6 +/-10.7 g (lower figure of body 3), and the body length of a normal individual (n ═ 200) is 15.5 +/-3.5 cm, and the body weight of the normal individual is 18.2 +/-5.1 g (upper figure of figure 3). The triploid Taiwan loach individual is significantly larger than the normal diploid individual, and the growth performance is outstanding (figure 3). By dissecting the gonads, the gonads of normal diploid individual-sized Taiwan loaches normally develop (lower panel of FIG. 4), while the gonads of triploid Taiwan loaches (including spermary and ovary) are dysplastic, the ovaries are linear, and mature ova are not seen (upper panel of FIG. 4).
Through statistics, the average body length of the triploid Taiwan loach is 20.3cm, the average weight of the triploid Taiwan loach is 88.6 g, the average body length of the normal diploid individual is 15.5cm, the average weight of the normal diploid individual is 18.2g, and the growth performance of the triploid Taiwan loach is outstanding. The gonad development of the triploid Taiwan loach is low, and gonad development energy or nutrition can be used for growth and meat quality improvement, so that the triploid Taiwan loach has advantages in growth.
The above embodiments are only used for further illustration of the present invention, and are not intended to limit the scope of the present invention, and all equivalent changes made based on the concept of the present invention and obvious modifications of various technical solutions of the present invention fall within the scope of the present invention.
Claims (5)
1. A method for cultivating triploid loaches is characterized by comprising the following steps: the cultivation method comprises the following steps:
(a) fertilizing the loach sperms and the loach ova to form fertilized ova;
(b) 3min after fertilization, treating the fertilized eggs for 3min by using hydrostatic shock of 40 MPa;
(c) hatching the fertilized eggs to obtain the triploid loaches;
the loach is selected from Taiwan loach.
2. A cultivation method as claimed in claim 1, characterised in that:
the loach sperm is obtained by squeezing the abdomen of a mature male loach.
3. A cultivation method as claimed in claim 1, characterised in that:
in the step (a), the loach ovum obtaining step is as follows:
(1) injecting 800-1200IU/kg bw of chorionic gonadotropin and 3-6 mu g/kg of luteinizing hormone releasing hormone analogue into the base part of the pectoral fin of the female loach;
(2) allowing the injected loaches to move in water for 6-12 hours;
(3) and extruding the belly of the loach to obtain the loach ovum.
4. A cultivation method as claimed in claim 1, characterised in that:
in step (a), the fertilization is a dry fertilization.
5. A cultivation method as claimed in claim 1, characterised in that:
the hydrostatic pressure operation steps are as follows: and mixing the activated loach fertilized eggs with water, placing the mixture in a pressure container, and applying hydrostatic pressure to the activated loach fertilized eggs and the water by applying mechanical pressure to the pressure container.
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Citations (3)
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|---|---|---|---|---|
| CN101940169A (en) * | 2010-07-02 | 2011-01-12 | 北京卧佛山庄养殖有限公司 | Rainbow trout tetraploid breeding method |
| CN102041661A (en) * | 2009-10-13 | 2011-05-04 | 三星电子株式会社 | Apparatus for controlling of door lock in washing machine and method thereof |
| CN104396812A (en) * | 2014-10-27 | 2015-03-11 | 华中农业大学 | Method for producing allopolyploid misgurnus anguillicaudatus through inhibiting fertilized egg polar body release |
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2019
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102041661A (en) * | 2009-10-13 | 2011-05-04 | 三星电子株式会社 | Apparatus for controlling of door lock in washing machine and method thereof |
| CN101940169A (en) * | 2010-07-02 | 2011-01-12 | 北京卧佛山庄养殖有限公司 | Rainbow trout tetraploid breeding method |
| CN104396812A (en) * | 2014-10-27 | 2015-03-11 | 华中农业大学 | Method for producing allopolyploid misgurnus anguillicaudatus through inhibiting fertilized egg polar body release |
Non-Patent Citations (2)
| Title |
|---|
| Production of Second Generation Progeny of Hexaploid Loach;ARAI K等;《Fisheries Science》;19991231;第65卷(第2期);第65卷第2期 * |
| 冷休克诱导泥鳅三倍体的研究;康绍乐等;《淡水渔业》;20081130;第38卷(第6期);第76-79页 * |
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