CN114287366A - Production method and application of sterile seawater medaka adult fish - Google Patents
Production method and application of sterile seawater medaka adult fish Download PDFInfo
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- CN114287366A CN114287366A CN202111460321.6A CN202111460321A CN114287366A CN 114287366 A CN114287366 A CN 114287366A CN 202111460321 A CN202111460321 A CN 202111460321A CN 114287366 A CN114287366 A CN 114287366A
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Abstract
The invention discloses a production method and application of a sterile adult medaka in seawater. The production method comprises the following steps: placing the fertilized eggs of the seawater medaka in sterile treatment liquid for culturing; then rinsing the fertilized eggs with a sterile culture solution and soaking the fertilized eggs in a bacteriostatic solution; and then rinsing the soaked fertilized eggs with a sterile culture solution, soaking the fertilized eggs in a bleaching solution for bleaching, rinsing the bleached fertilized eggs with the sterile culture solution, and placing the rinsed fertilized eggs in the sterile culture solution for culturing to obtain the sterile seawater medaka adult fish. Obtaining seawater medaka zygotes through natural propagation, obtaining sterile embryos through a series of antibiotic and chemical substance treatment, and finally placing the sterile seawater medaka zygotes in a sterile environment for culturing to obtain sterile seawater medaka adult fish; the invention provides convenient materials for researching the interaction between the microorganism and the medaka in seawater and the multi-biological process, and has important reference significance for further researching the internal mechanism of the relationship between the microorganism and the vertebrate host.
Description
Technical Field
The invention relates to the technical field of fish production, in particular to a production method and application of a sterile seawater medaka adult fish.
Background
The seawater medaka (Oryzias melastinma) belongs to phyla chordata, subphylum vertebrates, class Osteichthyes, class RADIX subphylum, order killifida, family medakaceae and genus medaka, also called medaka and Indian Mada, and is a marine fish with mild sexual behavior and omnivory. The ocean medaka is often used as a toxicology research model and has the advantages that: the method is easy for large-scale laboratory culture: adult fish is small in size, generally 2-4cm, obvious in sex difference and short in generation cycle, and is fed twice a day, wherein the light cycle is 14 hours under illumination and 10 hours in darkness; high spawning rate: all-year spawning, wherein the spawning peak time is 4-9 months, generally, spawning starts in the first hour of the start of a photoperiod every day, the spawning amount is 10-30 eggs/d, the fertilization rate exceeds 80%, sexual maturity can be achieved in 3-4 months after hatching, and the requirement on water quality is not high; the bearing capacity on salinity and temperature is very big: can adapt to different salinity environments, can tolerate the temperature of 7-30 ℃, and has the optimal temperature of about 25 ℃. In the first few weeks after fertilization, medaka lacks the adaptive immune system, and relies entirely on the innate immune system to address the immune challenge, and the development of tissues and organs involved in adaptive and humoral immunity can be observed for about 4-6 weeks.
The sterile medaka in seawater can be used for carrying out a sterility test, and the light transparency property of the medaka in seawater during development allows an experimenter to observe host and microbial cells in vivo, so that how the microbes are colonized or how metabolic products of the microbes influence the host biological processes, including gene expression, development, physiology, immunity and lifespan can be studied by exposing the sterile medaka in seawater to individual strains, specific microbes or products at specific time points and then analyzing the reaction of the host; the in vivo competition between microbial populations can also be detected by studying microbial interactions within the physiological environment of living hosts, using defined microbial species or combinations of genotypes to colonize the sterile hosts, and using culture or DNA sequences to detect in vivo competition between microbial populations, and the host-to-microbial and microbial-to-microbial interactions can be used as a function to analyze microbial genotypes, population competition, host genotypes, host developmental stages, host physiology, history of microbial colonization in the hosts, anatomical localization, diet and other environmental and physiological parameters.
Therefore, the cultivation of the sterile seawater medaka has important significance for the sterile animal experiment.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, the present invention aims to provide a method for producing a sterilized adult medaka and its use.
In order to achieve the above objects and other related objects, a first aspect of the present invention provides a method for producing a sterilized adult medaka, comprising the steps of:
(1) placing the in-vitro seawater medaka zygotes in sterile treatment solution for culturing;
(2) rinsing the fertilized eggs treated in the step (1) by using sterile culture solution;
(3) soaking the rinsed fertilized eggs in the step (2) in a bacteriostatic solution;
(4) rinsing the fertilized eggs soaked in the step (3) by using sterile culture solution;
(5) soaking the fertilized eggs rinsed in the step (4) in bleaching liquid for bleaching;
(6) rinsing the fertilized eggs bleached in the step (5) by using a sterile culture solution, and then placing the fertilized eggs into the sterile culture solution for culturing to obtain sterile seawater medaka adult fish.
Further, in the step (1), the sterile treatment solution is an AB-MMEM solution, the solvent of the AB-MMEM solution is water, and solutes and the concentrations thereof are as follows: 400-500 ng/mL amphotericin B, 5-10. mu.g/mL kanamycin, 100-150. mu.g/mL ampicillin, 10-20U/mL penicillin, 10-20U/mL streptomycin, and 30-35 mg/mL sea salt, preferably 500ng/mL amphotericin B, 10. mu.g/mL kanamycin, 100. mu.g/mL ampicillin, 10U/mL penicillin, 10U/mL streptomycin, and 35mg/mL sea salt.
Further, in the step (1), the culture temperature is 25 +/-1 ℃, and the culture time is 6-8 h.
Further, in the steps (2), (4) and (6), the sterile culture solution is sterile seawater, the sterile seawater is a sterile MMEM solution, a solvent of the sterile MMEM solution is RO water, and a solute of the sterile MMEM solution is sea salt; preferably, the concentration of the sterile MMEM solution is 30-35 g/L.
Further, in the steps (2), (4) and (6), the rinsing times are 2-3 times.
Further, in the step (3), the antibacterial liquid is povidone iodine aqueous solution; preferably, the concentration of the povidone-iodine aqueous solution is 0.2-0.3 g/L.
Further, in the step (3), the fertilized eggs are soaked in the bacteriostatic solution for 1-2 min.
Further, in the step (5), the bleaching solution is NaClO solution; preferably, the bleaching solution is NaClO solution with the concentration of 0.02-0.04 g/L.
Further, in the step (5), the fertilized eggs are soaked in the bleaching solution for 10-15 min.
Further, in the step (6), the culture density is 40-60 fertilized eggs are cultured in each 100mL of sterile culture solution.
Further, in the step (6), the culture temperature is 25. + -. 1 ℃, the photoperiod Lx: dy is L10: d14 to L14: d10, wherein the light period Lx: dy means light time x hours per day, dark time x hours per day, and x + y is 24.
Further, in the step (6), when the fertilized eggs are 7 days old, the whole amount of liquid exchange is carried out; and after the fertilized eggs are hatched into juvenile fish, performing full-scale liquid change culture of the sterile culture liquid on the juvenile fish every day until the juvenile fish grows into a seawater medaka of the target size, and then obtaining the target sterile seawater medaka from the seawater medaka of the target size.
Further, in the step (6), the method for obtaining the sterile medaka from the medaka of the target size comprises the following steps: and (3) performing sterility test on the sterile MMEM solution which is used for culturing the seawater medaka with the target size, wherein the seawater medaka with the target size cultured in the sterile MMEM solution with the test sterility is the sterile seawater medaka with the target size.
Further, after hatching the fertilized egg embryo into juvenile fish, the sterility of the obtained seawater medaka is examined every three days.
Further, the sterility test method is at least one selected from the group consisting of a TSA plate culture method, a TSB liquid culture method, an anaerobe TSB liquid assay method, and a 16sV3PCR assay method.
Further, the TSA plate culture method comprises the steps of: and uniformly coating the culture solution to be detected on a TSA flat plate, inverting the flat plate in an incubator at 30 ℃, culturing for one week under aerobic condition, observing whether the flat plate is infected with bacteria, determining that the seawater medaka bred by the culture solution is sterile seawater medaka if bacteria on the flat plate grow, and determining that the seawater medaka bred by the culture solution is not sterile if the bacteria on the flat plate grow.
Further, the TSB liquid culture method comprises the following steps: injecting the culture solution to be detected into the TSB liquid, culturing for a week under the aerobic condition in a shaker at 180rpm, observing whether the TSB liquid is contaminated, determining that the seawater medaka bred by the culture solution is sterile seawater medaka if the TSB liquid is clear, and determining that the seawater medaka bred by the culture solution is not sterile if the TSB liquid is turbid.
Further, the anaerobic TSB liquid culture method comprises the following steps: injecting the culture solution to be detected into anaerobic TSB liquid, culturing for a week in a shaker at 180rpm under anaerobic condition, observing whether the strain is contaminated, determining that the seawater medaka bred by the culture solution is sterile seawater medaka if the TSB liquid is clear, and determining that the seawater medaka bred by the culture solution is not sterile if the TSB liquid is turbid.
Further, the 16sV3PCR method comprises the steps of: and (3) performing PCR amplification by using the culture solution to be detected as a template and using a primer designed aiming at a section of conserved sequence in the bacterial 16S ribosomal DNAV3 area, wherein if the PCR amplification product has a target band with the size of 198bp, the medaka prepared by using the culture solution to be detected is not sterile, the experimental operation fails, and if the PCR amplification product does not have the target band with the size of 198bp, the medaka prepared by using the culture solution to be detected can be determined to be sterile.
Further, the method for producing the sterilized adult medaka fish in seawater further comprises the following steps: feeding the seawater medaka starting on the 3 rd day after the fertilized eggs are hatched.
Further, the feed is a sterile feed; preferably, the feed is sterile shrimp meal and/or sterile dehulled artemia.
Further, the preparation method of the sterile shrimp meal comprises the following steps: and (3) performing high-temperature high-pressure sterilization treatment on the shrimp powder particles at least twice to obtain the sterile shrimp powder.
Further, in the preparation method of the sterile shrimp meal, the high-temperature and high-pressure sterilization treatment conditions are as follows: 120 ℃, 30min and 103.4 kPa.
Further, the preparation method of the sterile hulled fairy shrimp comprises the following steps: and (3) performing high-temperature high-pressure treatment on the shelled brine shrimp eggs for at least two times to obtain sterile shelled brine shrimp.
Further, in the preparation method of the sterile hulled fairy shrimp, the high-temperature and high-pressure sterilization treatment conditions are as follows: 120 ℃, 30min and 103.4 kPa.
Further, the feeding amount is that 0.1mL of sterile shrimp meal solution or 20-30 sterile shelled fairy shrimps are added into each 3mL of sterile culture solution used in the process of breeding the seawater medaka, and the sterile shrimp meal solution is prepared by adding 0.08-0.12 g of sterile shrimp meal into 3mL of sterile culture solution and mixing.
Further, the feed was fed once a day.
Further, the method for producing the sterilized adult medaka fish in seawater further comprises the following steps: culturing male and female sexually mature medaka together, and naturally breeding to obtain medaka zygotes.
Further, sexually mature male and female medaka fish are cultured in a sterile culture solution.
Further, the fertilized eggs of the seawater medaka are rinsed for a plurality of times by using the AB-MMEM solution and then cultured by using the AB-MMEM solution.
The second aspect of the invention provides the use of the method according to the first aspect in producing a sterile adult medaka.
In a third aspect, the invention provides an AB-MMEM solution, the solvent of which is water, and the solutes and their concentrations are as follows: 400-500 ng/mL amphotericin B, 5-10 mug/mL kanamycin, 100-150 mug/mL ampicillin, 10-20U/mL penicillin, 10-20U/mL streptomycin and 30-35 mg/mL sea salt.
Further, the solutes and their concentrations of the AB-MMEM solution are as follows: amphotericin B at 500ng/mL, kanamycin at 10. mu.g/mL, ampicillin at 100. mu.g/mL, penicillin at 10U/mL, streptomycin at 10U/mL, sea salt at 35 mg/mL.
A fourth aspect of the invention provides the use of the AB-MMEM solution according to the third aspect in the production of a sterile medaka in seawater.
As described above, the production method and application of the sterilized adult medaka have the following beneficial effects:
the method obtains the medaka zygotes in seawater through natural propagation, obtains sterile embryos through a series of antibiotic and chemical substance treatment, and finally, places the medaka zygotes in a sterile environment for culture to finally obtain the sterile medaka in seawater. The invention provides convenient materials for researching the interaction between the microorganism and the medaka in seawater and the multi-biological process, and has very important reference significance for further researching the internal mechanism of the relationship between the microorganism and the vertebrate host.
Drawings
FIG. 1 shows a production flow chart of a sterilized adult medaka in seawater according to an embodiment of the present invention.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The experimental methods used in the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the present invention are commercially available unless otherwise specified.
In the present invention, the light period Lx: dy represents a light-on period of x hours, a dark period of y hours, and x + y is 24 per day.
The invention provides a method for producing a sterile adult medaka in seawater, which comprises the following steps:
(1) placing the in-vitro seawater medaka fertilized eggs into sterile treatment liquid for culturing at the culture temperature of 25 +/-1 ℃ for 6-8 h;
(2) rinsing the fertilized eggs treated in the step (1) with sterile culture solution for 2-3 times;
(3) soaking the fertilized eggs rinsed in the step (2) in a bacteriostatic solution for 1-2 min;
(4) rinsing the fertilized eggs soaked in the step (3) for 2-3 times by using sterile culture solution;
(5) soaking the fertilized eggs rinsed in the step (4) in bleaching liquid for bleaching, wherein the soaking time is 10-15 min;
(6) rinsing the fertilized eggs bleached in the step (5) for 2-3 times by using a sterile culture solution, and then placing the fertilized eggs into the sterile culture solution for culturing to obtain sterile seawater medaka adult fish, wherein the culture conditions and the process are as follows:
culturing 40-60 fertilized eggs in each 100mL sterile culture solution at the culture temperature of 25 +/-1 ℃ in a photoperiod Lx: dy is L10: d14 to L14: d10;
performing total liquid change when the fertilized eggs are 7 days old;
after the fertilized egg embryo is hatched into the juvenile fish, performing half-amount liquid changing culture of the sterile culture liquid on the juvenile fish every day, detecting the sterility of the obtained seawater medaka every three days until the juvenile fish grows into the seawater medaka of the target size, and then obtaining the target sterile seawater medaka from the seawater medaka of the target size;
feeding the seawater medaka once every day from 3 days after the fertilized eggs are hatched, wherein the feeding is sterile feed (sterile shrimp meal and/or sterile shelled artemia) and the feeding amount is 0.1mL of sterile shrimp meal solution or 20-30 sterile shelled artemia added in each 3mL of sterile culture solution used in the process of feeding the seawater medaka.
In the step (1), the sterile treatment solution is an AB-MMEM solution, the solvent of the AB-MMEM solution is water, and solutes and the concentrations thereof are as follows: 400-500 ng/mL amphotericin B, 5-10. mu.g/mL kanamycin, 100-150. mu.g/mL ampicillin, 10-20U/mL penicillin, 10-20U/mL streptomycin, and 30-35 mg/mL sea salt, preferably 500ng/mL amphotericin B, 10. mu.g/mL kanamycin, 100. mu.g/mL ampicillin, 10U/mL penicillin, 10U/mL streptomycin, and 35mg/mL sea salt.
In the steps (2), (4) and (6), the sterile culture solution is sterile seawater, the sterile seawater is a sterile MMEM solution, a solvent of the sterile MMEM solution is RO water, and a solute is sea salt; preferably, the concentration of the sterile MMEM solution is 30-35 g/L.
Wherein in the step (3), the antibacterial liquid is povidone iodine aqueous solution; preferably, the concentration of the povidone-iodine aqueous solution is 0.2-0.3 g/L.
Wherein, in the step (5), the bleaching solution is NaClO solution; preferably, the bleaching solution is NaClO solution with the concentration of 0.02-0.04 g/L.
Wherein the sterility test method is at least one selected from the group consisting of a TSA plate culture method, a TSB liquid culture method, an anaerobe TSB liquid detection method and a 16sV3PCR detection method.
The present invention will be described in detail with reference to the following specific examples. It should also be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention, and that numerous insubstantial modifications and adaptations of the invention described above will occur to those skilled in the art. The specific process parameters and the like of the following examples are also only one example of suitable ranges, i.e., those skilled in the art can select the appropriate ranges through the description herein, and are not limited to the specific values exemplified below.
Example 1
First, experimental material
Amphotericin B, kanamycin, ampicillin, penicillin and streptomycin mixtures were purchased from Solarbio.
Povidone iodine (a complex of polyvinylpyrrolidone and iodine) (PVP-I) was purchased from aladdin corporation.
Bleaching agent: mother liquor: sodium hypochlorite aqueous solution with the concentration of 10 g/L; working fluid: sodium hypochlorite aqueous solution with concentration of 0.04 g/L.
Sea salt: landebao (Landebao) activating salt.
The special feed for the zebra fish comprises the following components: shrimp meal, brand zeiglee, available from shanghai saint bio-equipment ltd; shelled fairy shrimp, brand red egg, purchased from shangjia aquarium.
Sterile MMEM solution: mixing 1L water with 35g sea salt, mixing well, and sterilizing at high temperature under high pressure. The content of sea salt in the MMEM solution is 35 mg/mL.
Amphotericin B stock (500ng/mL), kanamycin stock (10mg/mL), ampicillin stock (100. mu.g/mL), penicillin-streptomycin stock (10U/mL for both penicillin and streptomycin). The solvent of each stock solution is RO water.
AB-MMEM solution: 49.6mL of MMEM solution, 100. mu.L of amphotericin B stock solution, 50. mu.L of kanamycin stock solution, 250. mu.L of ampicillin stock solution, and 50. mu.L of penicillin-streptomycin stock solution were mixed, filtered through a 0.22 μm filter, and dispensed into 50mL conical tubes to be stored at-20 ℃. The content of amphotericin B in the AB-MMEM solution is 500ng/mL, the content of kanamycin is 10 mug/mL, the content of ampicillin is 100 mug/mL, the content of penicillin and streptomycin is 10U/mL respectively, and the content of sea salt is 35 mg/mL.
Production and effect evaluation of sterile seawater medaka
As shown in FIG. 1, the production process of the sterilized adult medaka comprises the following steps:
(1) two hours after feeding in the afternoon, three pairs of sexually mature marine medaka were placed in a clean crystallizing dish and cultured with a sterile MMEM solution.
(2) The next day, female fish lay eggs in natural state, male fish are fertilized in vitro, after fertilized eggs naturally fall off from the female fish cloacae, the fertilized eggs are immediately collected by a suction pipe, the adhered fertilized eggs are separated, the AB-MMEM solution is rinsed for three times, and the fertilized eggs are transferred to a cell culture dish filled with the AB-MMEM solution and cultured for 7h at 25 ℃ (prompting that the fertilized eggs can be cultured for 6-8 h).
(3) Removing low-quality fertilized eggs with white spots or whitening, and selecting high-quality fertilized eggs with transparent and complete protective films for the following operation, wherein the time is about 40 min.
(4) And (3) rinsing the fertilized eggs selected in the step (3) by using a sterile MMEM solution for 3 times (about 10min), and then gently immersing the fertilized eggs into a PVP-I aqueous solution with the concentration of 0.2g/L for 1 min. (Note: it cannot exceed 1.5min, otherwise the mortality of fertilized egg embryos is increased).
(5) And (3) rinsing the fertilized eggs treated in the step (4) by using a sterile MMEM solution for 3 times (about 10min), bleaching for 10min (10-15 min is prompted) by using a bleaching agent working solution (obtained by diluting a bleaching agent mother solution by using the MMEM solution) with the sodium hypochlorite concentration of 0.04g/L, and slightly shaking at intervals to suspend the fertilized eggs in the bleaching agent. Care was taken to minimize air bubbles in the solution.
(6) And (3) rinsing the fertilized eggs treated in the step (5) by using a sterile MMEM solution for 3 times (about 10min), then transferring the fertilized eggs into a 24-pore plate for culture, culturing 50 fertilized egg embryos in the MMEM solution with the culture density of 100mL, placing the fertilized eggs in a super clean bench or a biochemical incubator, and performing light cycle L14: and D10.
(7) And (3) performing full-scale liquid replacement when the medaka zygotes are 7 days old, namely removing all volumes of old culture solution, simultaneously supplementing equal-volume sterile MMEM solution, observing the development state of the medaka zygotes and counting the survival rate of the medaka zygotes, performing TSA plate culture on 100 mu L of culture solution, performing TSB liquid culture on 200 mu L of culture solution, performing anaerobic TSB culture on 200 mu L of culture solution, and performing 16sV3PCR on 10 mu L of culture solution to detect the sterility of the obtained medaka.
(8) And after 13-16 days, hatching the fertilized egg embryo into juvenile fish, and then carrying out full liquid change every day, namely removing all old culture solution with the same volume, and simultaneously supplementing sterile MMEM solution with the same volume.
(9) And after the fertilized egg embryo is hatched into juvenile fish, regularly observing the living state of the seawater medaka every day, counting the survival rate of the seawater medaka, and detecting the sterility of the obtained seawater medaka every three days.
Wherein the survival rate is the number of the seawater medaka surviving in observation/the number of the fertilized eggs selected in the step (3) multiplied by 100 percent;
the sterile rate is the number of sterile seawater medaka/the number of surviving seawater medaka multiplied by 100 percent.
The operation method for detecting the sterility of the medaka in seawater comprises the following steps:
TSA plate culture method: the culture solution to be tested was spread on a TSA plate, which was then inverted and cultured at 30 ℃ for one week under aerobic conditions, and the plate was observed for the presence of colonies. And a result judgment method comprises the following steps: if colonies grow on the plate, taking out the medaka in seawater fed with the measured culture solution if the medaka in seawater is not sterile; if no colony grows on the plate, the medaka bred with the measured culture solution is determined to be a sterile medaka. Wherein the TSA plate is prepared from 6g of tryptone, 2g of soybean peptone, 2g of NaCl, 6g of agar and 400mL of RO water by shaking, uniformly mixing, sterilizing at high temperature and high pressure, inverting the plate, and cooling.
TSB liquid culture method: the culture solution to be tested is injected into the TSB liquid, cultured in a shaker at 180rpm for one week at 30 ℃ under the aerobic condition, and whether the TSB liquid is turbid or not is observed. And a result judgment method comprises the following steps: if the TSB liquid is turbid, taking out the medaka in seawater with bacteria, wherein the medaka in seawater bred by the measured culture solution is not sterile; and if the TSB liquid is clear, determining that the medaka bred by the measured culture solution is sterile medaka. Wherein the TSB liquid is prepared by shaking and uniformly mixing 6g of tryptone, 2g of soybean peptone, 2g of NaCl and 400mLRO water, sterilizing at high temperature and high pressure, and cooling.
Anaerobic TSB liquid culture method: and (3) injecting the culture solution to be detected into the anaerobic TSB liquid, culturing for a week in a shaker at 180rpm at 30 ℃ under an anaerobic condition, and observing whether the TSB liquid is turbid or not. And a result judgment method comprises the following steps: if the TSB liquid is turbid, taking out the medaka in seawater with bacteria, wherein the medaka in seawater bred by the measured culture solution is not sterile; and if the TSB liquid is clear, determining that the medaka bred by the measured culture solution is sterile medaka. The TSB liquid is 6g of tryptone, 2g of soybean peptone, 2g of NaCl and 400mL of RO water, is shaken and uniformly mixed, then oxygen is removed, and then the TSB liquid is sterilized at high temperature and high pressure and can be used after being cooled.
16sV3 PCR: the principle for detecting the sterility of the medaka in seawater comprises the following steps: a primer is designed according to a conservative sequence of the bacterium in a 16S ribosomal DNA V3 area for PCR amplification, and the amplification fragment is 198 bp. The detection method comprises the following steps: the presence of any bacteria in the vessel was detected using the 16s ribosomal RNA target gene using the culture to be tested as a template. The reaction system is as follows: 2.5. mu.L of 10xPCR buffer, 2. mu.L of dNTPmix (2.5mmol/L), 0.5. mu.L of primer 338F (20. mu. mol/L), 0.5. mu.L of primer 519R (20. mu. mol/L), 0.5. mu.L of rTaq (2.5U/. mu.L), 10. mu.L of template, and 20. mu.L of 4. mu.L water in total. The PCR reaction program is: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 53 ℃ for 30s, 72 ℃ for 30 s; extension at 72 ℃ for 10 min. And a result judgment method comprises the following steps: if the PCR amplification product has a target band with the size of 198bp, the seawater medaka bred by the culture solution to be tested is not sterile and is removed; and if the PCR amplification product does not have a band with the size of 198bp, determining that the seawater medaka bred by the culture solution to be tested is sterile seawater medaka.
338F：
5’-CGCCCGCCGCGCGCGGCGGCGGGCGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3’；
519R：
5’-ATTACCGCGGCTGCTGG-3’。
(10) Feeding the young medaka in seawater on the 3 rd day after hatching. Feeding once a day. Each feeding was performed as follows: the feeding amount of each time is that 0.1mL of sterile shrimp meal solution (prepared by adding 0.08-0.12 g of sterile shrimp meal into 3mL of sterile culture solution and fully mixing) is added into every 3mL of MMEM solution (MMEM solution used in the process of feeding the seawater medaka) or 20-30 sterile hulled artemia.
The operation method of the sterile shrimp meal and the sterile unshelled fairy shrimp comprises the following steps:
the shrimp meal and the shelled fairy shrimp are respectively subpackaged in 0.2mL EP tubes and are sterilized at high temperature and high pressure twice (120 ℃, 30min, 103.4 kPa). And (3) verifying the sterility of the shrimp meal and the shelled fairy shrimp by a TSA plate culture method, a TSB liquid culture method and an anaerobic TSB liquid culture method, wherein the specific method and the result judgment standard are described in the step (9) in a related way.
With reference to the above method, three experiments were carried out (50 fertilized eggs were selected in the step (3) of each experiment) in total, and the results were averaged over three replicates.
Statistics of the survival rate and the sterile rate of the medaka in seawater 1 to 30 days (1 to 30dpf) after the fertilized egg embryo is hatched into the juvenile fish in three repeated experiments are shown in table 1.
TABLE 1 statistics of three-time repeated experiments on the survival rate and the sterile rate of medaka in seawater at 1-30dpf
As can be seen from Table 1, the survival rate of the sterile seawater medaka is more than 25% from 1 to 30 days after the fertilized egg embryo is hatched into juvenile fish.
Therefore, the sterile medaka prepared from seawater can be simply and quickly obtained by the production method, so that very important materials, such as disease treatment mechanisms of pathogenic bacteria, basic researches such as probiotic effects of probiotics and biomedical researches, can be provided for researching the interaction between the microorganisms and the host.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
SEQUENCE LISTING
<110> Chongqing Mian Kai Biotech research institute Co., Ltd
<120> production method and application of sterilized adult medaka in seawater
<130> PYZMK2118003-HZ
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<170> PatentIn version 3.5
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attaccgcgg ctgctgg 17
Claims (10)
1. A method for producing a sterile adult medaka fish, which is characterized by comprising the following steps:
(1) placing the in-vitro seawater medaka zygotes in sterile treatment solution for culturing;
(2) rinsing the fertilized eggs treated in the step (1) by using sterile culture solution;
(3) soaking the rinsed fertilized eggs in the step (2) in a bacteriostatic solution;
(4) rinsing the fertilized eggs soaked in the step (3) by using sterile culture solution;
(5) soaking the fertilized eggs rinsed in the step (4) in bleaching liquid for bleaching;
(6) rinsing the fertilized eggs bleached in the step (5) by using a sterile culture solution, and then placing the fertilized eggs into the sterile culture solution for culturing to obtain sterile seawater medaka adult fish.
2. The production method according to claim 1, characterized in that: in the step (1), the sterile treatment solution is an AB-MMEM solution, the solvent of the AB-MMEM solution is water, and solutes and the concentrations thereof are as follows: 400-500 ng/mL amphotericin B, 5-10 ug/mL kanamycin, 100-150 ug/mL ampicillin, 10-20U/mL penicillin, 10-20U/mL streptomycin and 30-35 mg/mL sea salt;
and/or in the step (1), the culture temperature is 25 +/-1 ℃, and the culture time is 6-8 h;
and/or in the steps (2), (4) and (6), the sterile culture solution is sterile seawater, the sterile seawater is a sterile MMEM solution, the solvent of the sterile MMEM solution is RO water, and the solute is sea salt;
and/or in the steps (2), (4) and (6), the rinsing times are 2-3 times;
and/or, in the step (3), the antibacterial liquid is povidone iodine aqueous solution;
and/or, in the step (3), soaking the fertilized eggs in the antibacterial solution for 1-2 min;
and/or, in the step (5), the bleaching solution is NaClO solution;
and/or, in the step (5), soaking the fertilized eggs in the bleaching solution for 10-15 min;
and/or, in the step (6), culturing 40-60 fertilized eggs in each 100mL of sterile culture solution at the culture density;
and/or, in the step (6), the culture temperature is 25 +/-1 ℃, the photoperiod Lx: dy is L10: d14 to L14:
d10, wherein the light period Lx: dy means light time x hours per day, dark time x hours per day, and x + y is 24.
3. The production method according to claim 2, characterized in that: the concentration of the sterile MMEM solution is 30-35 g/L;
the concentration of the povidone iodine aqueous solution is 0.2-0.3 g/L;
the bleaching solution is NaClO solution with the concentration of 0.02-0.04 g/L.
4. The production method according to claim 1, characterized in that: in the step (6), when the fertilized eggs are 7 days old, the whole amount of liquid exchange is carried out; after the fertilized eggs are hatched into juvenile fish, performing sterile seawater full-scale liquid change culture on the juvenile fish every day until the juvenile fish grows into seawater medaka of a target size, and then obtaining the target sterile seawater medaka from the seawater medaka of the target size;
and/or the production method of the sterile adult medaka comprises the following steps: feeding the seawater medaka on the 3 rd day after the fertilized eggs are hatched;
and/or the production method of the sterile adult medaka comprises the following steps: culturing male and female sexually mature medaka together, and naturally breeding to obtain medaka zygotes.
5. The production method according to claim 4, characterized in that: in the step (6), the method for obtaining the sterile medaka from the medaka of the target size comprises the following steps: performing sterility test on the sterile MMEM solution used for culturing the seawater medaka of the target size, wherein the seawater medaka of the target size cultured in the sterile MMEM solution subjected to sterility test is the sterile seawater medaka of the target size;
after the fertilized egg embryo is hatched into juvenile fish, detecting the sterility of the obtained seawater medaka every three days;
the feed is aseptic feed; feeding once a day;
culturing sexually mature male and female medaka fish with sterile culture solution.
6. The production method according to claim 5, characterized in that: the sterility detection method is at least one selected from a TSA plate culture method, a TSB liquid culture method, an anaerobe TSB liquid detection method and a 16sV3PCR detection method;
and feeding food, namely sterile shrimp meal and/or sterile shelled artemia, wherein the feeding amount is that 0.1mL of sterile shrimp meal solution or 20-30 sterile shelled artemia is added into every 3mL of sterile culture solution used in the process of breeding the seawater medaka.
7. The production method according to claim 6, characterized in that: the preparation method of the sterile shrimp meal comprises the following steps: performing high-temperature high-pressure sterilization treatment on the shrimp powder particles at least twice to obtain sterile shrimp powder;
the preparation method of the sterile hulled fairy shrimp comprises the following steps: and (3) performing high-temperature high-pressure treatment on the shelled brine shrimp eggs for at least two times to obtain sterile shelled brine shrimp.
8. Use of the method according to any one of claims 1 to 7 in the production of a sterile adult medaka.
9. An AB-MMEM solution characterized by: the solvent of the AB-MMEM solution is water, and the solutes and the concentrations thereof are as follows: 400-500 ng/mL amphotericin B, 5-10 mug/mL kanamycin, 100-150 mug/mL ampicillin, 10-20U/mL penicillin, 10-20U/mL streptomycin and 30-35 mg/mL sea salt.
10. Use of the AB-MMEM solution according to claim 9 in the production of a sterile medaka in seawater.
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| CN114680083A (en) * | 2022-05-05 | 2022-07-01 | 重庆医科大学 | Preparation method and application of sterile fairy shrimp |
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