In-vitro culture method for freshwater shellfish embryos

Abstract

The invention discloses a freshwater shellfish embryo in-vitro culture method, and belongs to the technical field of freshwater shellfish breeding. The method comprises the following steps that an antibiotic solution is prepared to serve as a disinfection reagent, shellfish and tools are disinfected in the antibiotic solution, and the antibiotic solution comprises sterile water, 50-80 [mu] g / mL of penicillin and 2-4 [mu] g / mL of amphotericin B; shellfish blood is taken and centrifuged for 3-6 min at the rotating speed of 1500-2500 r / min, and supernate is taken and is serum; mature sperms and egg cells are taken from the shellfish after blood taking to be prepared into a sperm solution and an egg cell solution, the sperm solution and the egg cell solution are mixed and then subjected to standing fertilization, and fertilized eggs are obtained; the fertilized eggs are placed in a culture medium to be cultured under the sterile condition of 21-23 DEG C, and the culture medium comprises serum and an antibiotic solution according to the volume ratio of (3-5): 1. The method is convenient to operate and low in cost, and fills the technical blank of in-vitro culture of freshwater shellfish embryos.

Description

Technical Field

[0001] The invention belongs to the technical field of freshwater shellfish breeding, and in particular relates to an in vitro culture method for freshwater shellfish embryos. Background Art

[0002] Freshwater shellfish have important economic (such as food, pearl cultivation) and ecological (such as water purification, carbon sink) values. However, freshwater shellfish have not yet achieved full artificial breeding. The most critical bottleneck lies in the particularity of their embryonic development: the fertilization method is between internal and external fertilization (the female clam transports the egg cells to the external gill cavity, the male clam discharges the sperm into the water, and the female clam absorbs the sperm into the external gill cavity by filtering water and fertilizes it here), and the fertilized egg needs to complete embryonic development in the external gill cavity of the female clam (including the cleavage stage, mulberry stage, blastocyst stage, gastrula stage and hook larvae stage). This severely limits the control range of artificial breeding. At the same time, it also greatly restricts the research and development and application of modern breeding technologies (such as polyploid induction and gene editing).

[0003] In view of this, previous researchers have attempted to carry out in vitro culture of freshwater shellfish embryos. Yu Ying et al. (Yu Ying, Hong Yijiang, Qiu Qijun, Wang Junhua, Xu Maoxi, Li Yunjuan. Observation on embryonic development and seasonal reproductive glands of the pond butterfly clam. Journal of Zoology, 2008, 43(3): 102-107) used a self-prepared balanced salt solution (formula: NaCl 0.503 g, KCl 0.051 g, Na2SO4 0.235 g, CaCl2 0.350 g, MgSO4·7H2O 0.090 g, NaHCO3 0.750 g, NaH2PO4 0.014 g, glucose 0.030 g per liter) to culture fertilized eggs from the external gill cavity of the pond butterfly clam in vitro. The embryonic development lasted for 22.5 h until the mulberry stage, after which development stopped. Hu Honghui et al. (Hu Honghui, Bai Zhiyi, Guo Sipeng, Feng Shangle, Sun Tianyang, Chen Weimin. Observation on early embryonic development of Hyriopsis cumbrina under in vitro culture conditions and analysis of zero-degree and effective accumulated temperature of embryonic development biology. Journal of Fisheries of China, 2021, 45(11): 1835−1842) obtained sperm and oocytes of Hyriopsis cumbrina by dissection, and then artificially inseminated them. The fertilized eggs were cultured in vitro at 25 ± 1 ℃ using the above-mentioned balanced salt solution. The embryonic development lasted up to 17.3 h and stopped after the mulberry stage. The reasons for the failure of embryonic culture in vitro of freshwater shellfish by previous researchers may be: (1) the nutrients in the culture medium are insufficient to support the development of the embryo; (2) the embryo is infected by bacteria / fungus during development. Summary of the invention

[0004] Technical problems solved: In response to the above technical problems, the present invention provides a method for in vitro culture of freshwater shellfish embryos, which is easy to operate and low-cost, filling the technical gap in in vitro culture of freshwater shellfish embryos.

[0005] Technical solution: A method for in vitro culture of freshwater shellfish embryos, comprising the following steps: S1. Disinfection of shellfish and tools: Prepare an antibiotic solution as a disinfectant, and disinfect shellfish and tools in the antibiotic solution, wherein the antibiotic solution includes sterile water, 50-80 μg / mL penicillin and 2-4 μg / mL amphotericin B; S2. Serum preparation: Take shellfish blood and centrifuge it at 1500-2500 r / min for 3-6 min. The supernatant is serum. S3. Artificial insemination: Mature sperm and egg cells are collected from the shellfish after bleeding to prepare sperm solution and egg cell solution, which are then mixed and left to stand for fertilization to obtain fertilized eggs; S4. In vitro embryo culture: The fertilized eggs are cultured in a culture medium under sterile conditions at 21-23°C, wherein the culture medium includes serum and antibiotic solution in a volume ratio of (3-5):1.

[0006] Preferably, the shellfish is the Odontostomus dorsalis or the Hylocereus triangularis clam.

[0007] Preferably, S1 to S4 are all performed under sterile conditions.

[0008] Preferably, the shellfish in S1 are in a sexually mature state, and the ratio of male to female is (3-5):1.

[0009] Preferably, the time for disinfecting shellfish and tools in S1 is 10 to 30 minutes.

[0010] Preferably, the method for collecting shellfish blood in S2 is: under sterile conditions, opening the shell with a shell opener, and extracting blood from the posterior adductor muscle venous sinus with a sterile syringe.

[0011] Preferably, the method for obtaining the egg cells in S3 is: under sterile conditions, cutting the adductor muscle of the female oyster after blood drawing with surgical scissors, transecting the gonad with a scalpel, scraping the gonad tissue with a thickness of 300-1000 μm, transferring it into sterile water and blowing it with a sterile pipette, and filtering out tissue blocks and immature egg cells with 150-250 μm and 50-70 μm sterile cell sieves respectively, thereby obtaining mature egg cells.

[0012] Preferably, the method for obtaining sperm in S3 is: under sterile conditions, cutting the adductor muscle of the male clam after blood drawing with surgical scissors, transecting the gonad with a scalpel, scraping the gonad tissue with a thickness of 300-1000 μm, transferring it into sterile water and blowing it with a sterile pipette, filtering out tissue blocks with a 50-70 μm sterile cell sieve, thereby obtaining mature sperm.

[0013] Preferably, the specific method of artificial insemination in S3 is: prepare an egg cell solution of 500-1000 egg cells / mL and a sperm solution of 2500-5000 sperm / mL in sterile water, respectively, mix the sperm solution and the egg cell solution in a volume ratio of 1:(3-5), let stand for fertilization, filter out the remaining sperm with a 50-70 μm sterile cell sieve, and obtain a fertilized egg.

[0014] Preferably, the static fertilization time in S3 is 5 to 20 minutes.

[0015] Beneficial effects: The present invention fills the gap in the in vitro culture technology of freshwater shellfish embryos. The original culture method has the significant advantages of convenient operation and low cost. The selected culture medium has a simple formula (consisting only of shellfish serum and antibiotic solution), and the shellfish serum comes from the same individual from which sperm and egg cells are taken, which not only makes full use of shellfish, but also avoids the expensive L15 culture medium commonly used in cell culture. DETAILED DESCRIPTION

[0016] The present invention will be further described below in conjunction with specific embodiments. Example 1

[0017] A method for culturing freshwater shellfish embryos in vitro comprises the following steps: 1. Prepare antibiotic solution: Prepare 5 L of antibiotic solution by adding 0.25 g of penicillin (50 μg / ml) and 0.01 g of amphotericin B (2 μg / ml) to 5 L of sterile water.

[0018] 2. Shellfish disinfection: One sexually mature male Odontodon dorsalis and three sexually mature female clams were selected and placed in glass tanks containing 2 L of antibiotic solution for disinfection for 10 min.

[0019] 3. Tool disinfection: Soak tools such as shell openers, surgical scissors, and scalpels in 1 L of antibiotic solution for 10 min for disinfection.

[0020] 4. Serum preparation: Under sterile conditions, the shells of female clams were opened with a shell opener, and 5 mL of blood was drawn from the venous sinus of the posterior adductor muscles of male and female clams with a sterile syringe. The blood was immediately centrifuged at 2000 r / min for 5 min, and the supernatant was the serum.

[0021] 5. Artificial insemination: Under sterile conditions, the adductor muscle of the female oyster after blood was drawn was cut with surgical scissors, the gonad was transversely cut with a scalpel, and the gonad tissue was scraped with a thickness of 300 μm, immediately transferred into sterile water and gently blown away with a sterile pipette, and the tissue blocks and immature oocytes were filtered out with 200 μm and 70 μm sterile cell sieves respectively to obtain mature oocytes; then the obtained mature oocytes were placed in sterile water, and the oocyte solution was adjusted to a density of 500 oocytes / mL.

[0022] Under sterile conditions, the adductor muscle of the male clams from which blood had been drawn was cut with surgical scissors, the gonad was transversely cut with a scalpel, and the gonadal tissue was scraped with a thickness of 300 μm. The solution was immediately transferred into sterile water and gently blown apart with a sterile pipette. The tissue blocks were filtered out with a 70 μm sterile cell sieve to obtain a mature sperm solution, and its density was adjusted to 2500 sperm / mL.

[0023] Under sterile conditions, the sperm solution and egg cell solution were mixed in a volume ratio of 1:3, and gently mixed with a sterile pipette, and allowed to stand for 10 min for full fertilization; then, the remaining sperm was filtered out with a 70 μm sterile cell sieve to obtain the fertilized egg.

[0024] 6. Embryo in vitro culture Under sterile conditions, 3 mL of mussel serum and 1 mL of antibiotic solution (i.e., the volume ratio of serum to antibiotic solution was 3:1) were used to prepare the culture medium; 4 mL of culture medium was added to the sterile culture dish, followed by 1000 fertilized eggs, and cultured at 21 ° C. After 20 days, the fertilized eggs successfully developed to the larval stage of the hook shell through the cleavage stage, mulberry stage, blastocyst stage and gastrula stage. Example 2

[0025] A method for culturing freshwater shellfish embryos in vitro comprises the following steps: 1. Prepare antibiotic solution: Prepare 10 L of antibiotic solution by adding 0.8 g of penicillin (80 μg / ml) and 0.04 g of amphotericin B (4 μg / ml) to 10 L of sterile water.

[0026] 2. Shellfish disinfection: Three sexually mature male clams of Odontodon dorsalis and 15 sexually mature female clams were selected. The male and female clams were placed in glass tanks containing 2 L and 7 L of antibiotic solution, respectively, and disinfected for 30 min.

[0027] 3. Tool disinfection: Soak shell openers, surgical scissors, and scalpels in 1 L of antibiotic solution for 30 min for disinfection.

[0028] 4. Serum preparation: Under sterile conditions, the shells of female clams were opened with a shell opener, and 10 mL of blood was drawn from the venous sinus of the posterior adductor muscles of male and female clams with a sterile syringe. The blood was immediately centrifuged at 2500 r / min for 6 min, and the supernatant was the serum.

[0029] 5. Artificial insemination: Under sterile conditions, the adductor muscle of the female clams after blood was drawn was cut with surgical scissors, the gonad was transversely cut with a scalpel, and the gonad tissue was scraped with a thickness of 1000 μm, immediately transferred into sterile water and gently blown away with a sterile pipette, and the tissue blocks and immature oocytes were filtered out with 250 μm and 70 μm sterile cell sieves respectively to obtain mature oocytes; then the mature oocytes were placed in sterile water, and the oocyte solution was adjusted to a density of 1000 oocytes / mL.

[0030] Under sterile conditions, the adductor muscle of the male clams from which blood had been drawn was cut with surgical scissors, the gonad was transversely cut with a scalpel, and the gonadal tissue was scraped with a thickness of 1000 μm. The solution was immediately transferred into sterile water and gently blown apart with a sterile pipette. The tissue blocks were filtered out with a 70 μm sterile cell sieve to obtain a mature sperm solution, and its density was adjusted to 5000 sperm / mL.

[0031] Under sterile conditions, the sperm solution and egg cell solution were mixed in a volume ratio of 1:4, and gently mixed with a sterile pipette, and allowed to stand for 20 min for full fertilization; then, the remaining sperm was filtered out with a 70 μm sterile cell sieve to obtain the fertilized egg.

[0032] 6. Embryo in vitro culture Under sterile conditions, 30 mL of mussel serum and 6 mL of antibiotic solution (i.e., the volume ratio of serum to antibiotic solution was 5:1) were used to prepare the culture medium; 10 mL of culture medium was added to three sterile culture dishes respectively, and then 2000 fertilized eggs were added respectively and cultured at 23 °C. After 19 days, the fertilized eggs successfully developed to the larval stage of the hook shell through the cleavage stage, mulberry stage, blastocyst stage and gastrula stage. Example 3

[0033] A method for culturing freshwater shellfish embryos in vitro comprises the following steps: 1. Prepare antibiotic solution: Prepare 5 L of antibiotic solution by adding 0.25 g of penicillin (50 μg / ml) and 0.01 g of amphotericin B (2 μg / ml) to 5 L of sterile water.

[0034] 2. Shellfish disinfection: One sexually mature male Hyriopsis cumingii and three sexually mature female Hyriopsis cumingii were selected, and the male and female clams were placed in glass tanks containing 2 L of antibiotic solution and disinfected for 10 min.

[0035] 3. Tool disinfection: Soak tools such as shell openers, surgical scissors, and scalpels in 1 L of antibiotic solution for 10 min for disinfection.

[0036] 4. Serum preparation: Under sterile conditions, the shells of female clams were opened with a shell opener, and 5 mL of blood was drawn from the venous sinus of the posterior adductor muscles of male and female clams with a sterile syringe. The blood was immediately centrifuged at 2000 r / min for 5 min, and the supernatant was the serum.

[0037] 5. Artificial insemination: Under sterile conditions, the adductor muscle of the female oyster after blood was drawn was cut with surgical scissors, the gonad was transversely cut with a scalpel, and the gonad tissue was scraped with a thickness of 300 μm, immediately transferred into sterile water and gently blown away with a sterile pipette, and the tissue blocks and immature oocytes were filtered out with 200 μm and 70 μm sterile cell sieves respectively to obtain mature oocytes; then the obtained mature oocytes were placed in sterile water, and the oocyte solution was adjusted to a density of 500 oocytes / mL.

[0038] Under sterile conditions, the adductor muscle of the male clams from which blood had been drawn was cut with surgical scissors, the gonad was transversely cut with a scalpel, and the gonadal tissue was scraped with a thickness of 300 μm. The solution was immediately transferred into sterile water and gently blown apart with a sterile pipette. The tissue blocks were filtered out with a 70 μm sterile cell sieve to obtain a mature sperm solution, and its density was adjusted to 2500 sperm / mL.

[0039] Under sterile conditions, the sperm solution and egg cell solution were mixed in a volume ratio of 1:3, and gently mixed with a sterile pipette, and allowed to stand for 10 min for full fertilization; then, the remaining sperm was filtered out with a 70 μm sterile cell sieve to obtain the fertilized egg.

[0040] 6. Embryo in vitro culture Under sterile conditions, 3 mL of mussel serum and 1 mL of antibiotic solution (i.e., the volume ratio of serum to antibiotic solution was 3:1) were used to prepare the culture medium; 4 mL of culture medium was added to the sterile culture dish, followed by 1000 fertilized eggs, and cultured at 21 ° C. After 20 days, the fertilized eggs successfully developed to the larval stage of the hook shell through the cleavage stage, mulberry stage, blastocyst stage and gastrula stage.

[0041] Comparative Example 4

[0042] When culturing embryos in vitro, five culture media were prepared under sterile conditions according to the embryonic development biology and environmental characteristics of the Odontodon dorsalis and the Hyriopsis cumingii: Medium I: A mixture of sterile water and antibiotic solution, with a volume ratio of 3 to 5:1 Medium II: A mixture of tap water filtered through a 0.22 μm sterile filter and aerated for 72 h and antibiotic solution. The volume ratio of tap water to antibiotic solution is 3-5:1. Medium III: A mixture of L15 medium and antibiotic solution. The volume ratio of L15 medium to antibiotic solution is 3-5:1. Medium IV: A mixture of serum from the Odontodon dorsalis or Hyriopsis cumingii and antibiotic solution, with the volume ratio of serum to antibiotic solution being 3-5:1 Medium V: A mixture of L15 medium, mussel serum and antibiotic solution. The volume ratio of L15 medium, serum and antibiotic solution is (1-2): (1-2): 1 Add 4-10 mL of each of the above five culture media to a sterile culture dish, then add 1000-2000 fertilized eggs, and culture at 21-23 °C under sterile conditions. During this period, examine the embryonic development under a microscope every 1-2 days, and replace the culture medium every 2-5 days.

[0043] The results showed that medium I, medium II and medium III were all unable to complete embryonic development, while medium IV and medium V were able to complete embryonic development, and there was no significant difference in the culture effects between medium IV and medium V. Considering the convenience of operation and cost, medium IV was better, that is, the medium formula selected in this application was better.

Claims

1. A method for culturing freshwater shellfish embryos in vitro, characterized in that: The steps include: S1. Disinfection of shellfish and tools: Prepare an antibiotic solution as a disinfectant, and disinfect shellfish and tools in the antibiotic solution, wherein the antibiotic solution includes sterile water, 50-80 μg / mL penicillin and 2-4 μg / mL amphotericin B; S2. Serum preparation: Take shellfish blood and centrifuge it at 1500-2500 r / min for 3-6 min. The supernatant is serum. S3. Artificial insemination: Mature sperm and egg cells are collected from the shellfish after bleeding to prepare sperm solution and egg cell solution, which are then mixed and left to stand for fertilization to obtain fertilized eggs; S4. In vitro embryo culture: The fertilized eggs are cultured in a culture medium under sterile conditions at 21-23°C, wherein the culture medium includes serum and antibiotic solution in a volume ratio of (3-5):

1.

2. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The shellfish is the dorsal horn toothless clam or the triangular sail clam.

3. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The steps S1 to S4 are all performed under sterile conditions.

4. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The shellfish in S1 are in a sexually mature state, and the ratio of male to female is (3-5):

1.

5. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The time for sterilizing shellfish and tools in S1 is 10 to 30 minutes.

6. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The method for collecting shellfish blood in S2 is as follows: under sterile conditions, the shell is opened with a shell opener, and blood is extracted from the posterior adductor muscle venous sinus with a sterile syringe.

7. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The method for obtaining the egg cells in S3 is as follows: under sterile conditions, the adductor muscle of the female oyster after blood drawing is cut with surgical scissors, the gonad is transversely cut with a scalpel, the gonad tissue is scraped with a thickness of 300-1000 μm, transferred into sterile water and blown away with a sterile pipette, and the tissue blocks and immature egg cells are filtered out with 150-250 μm and 50-70 μm sterile cell sieves respectively, so as to obtain mature egg cells.

8. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The method for obtaining sperm in S3 is as follows: under sterile conditions, the adductor muscle of the male clam after blood drawing is cut with surgical scissors, the gonad is transversely cut with a scalpel, the gonad tissue is scraped with a thickness of 300-1000 μm, transferred into sterile water and blown with a sterile pipette, and the tissue blocks are filtered out with a 50-70 μm sterile cell sieve to obtain mature sperm.

9. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The specific method of artificial insemination in S3 is: prepare an egg cell solution of 500-1000 egg cells / mL and a sperm solution of 2500-5000 sperm cells / mL in sterile water, respectively, mix the sperm solution and the egg cell solution in a volume ratio of 1:(3-5), let it stand for fertilization, filter out the remaining sperm with a 50-70 μm sterile cell sieve, and obtain a fertilized egg.

10. The in vitro culture method for freshwater shellfish embryos according to claim 1, characterized in that: The static fertilization time in S3 is 5 to 20 minutes.