Culture medium for in-vitro induced differentiation of paralichthys olivaceus spermatogonial stem cells and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to a culture medium for in-vitro induced differentiation of paralichthys olivaceus spermatogonial stem cells and application. According to the method, by optimizing an induced differentiation culture medium formula, combining specific growth factors and hormones and establishing a step-by-step collection and artificial insemination procedure, directional differentiation from long-term passage spermatogonial stem cells to sperms with insemination ability is successfully realized, and the paralichthys olivaceus offspring which normally develops is finally obtained. The method breaks through the key technical bottleneck that the sperm function is incomplete after induced differentiation of long-term passage spermatogonial stem cells, and survival offspring cannot be obtained, and provides a reliable technical platform for bastard halibut germplasm resource preservation, genetic breeding acceleration and reproductive development research.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a culture medium for in vitro induction of differentiation of turbot spermatogonial stem cells and its application. Background Technology

[0002] Fish spermatogonial stem cells are male reproductive stem cells capable of passing on genetic resources to the next generation. Once a spermatogonial stem cell line is established, a large number of reproductive stem cells can be obtained within a certain period. In vitro induced differentiation technology and reproductive stem cell transplantation technology can then be used to achieve the efficient preservation of male fish germplasm resources. However, reproductive stem cell transplantation technology has certain limitations, including the need for time to prepare infertile recipients, a long time from recipient to reproductive stage, risks such as disease, and high costs associated with surrogate recipient preservation. Once in vitro induced differentiation technology is mastered, the process from spermatogonial stem cells to sperm can be achieved within a short period of one month in the laboratory, eliminating the need for a full reproductive cycle as in vivo. This significantly accelerates the developmental process, providing a research platform for the molecular regulatory mechanisms of reproductive cell development and offering strong technical support for the protection of fish germplasm resources and even accelerating the breeding cycle.

[0003] Regarding the induction and differentiation of spermatogonial stem cells in fish, aquaculture experts have conducted in vitro studies on the induction and differentiation of primary spermatogonial stem cells from zebrafish, catfish, dark-lipped barbel, Japanese eel, and Chinese goby. However, the methods used for in vitro induction and differentiation, sperm collection, and artificial insemination are not suitable for long-term passage of spermatogonial stem cell lines, and cannot produce viable offspring from these lines. Furthermore, while long-term passaged spermatogonial cell lines from medaka, chub, grouper, redfin pufferfish, American shad, anchovy, and largemouth catfish produced sperm-like cells through induction and differentiation, they failed to produce viable offspring. Summary of the Invention

[0004] Based on this need, this invention proposes a culture medium for in vitro induction of differentiation of turbot spermatogonial stem cells and its related applications. It can stably and easily carry out in vitro induction of differentiation, collection and artificial insemination of turbot spermatogonial stem cells through long-term passage, and effectively solves the problem that turbot cannot produce offspring after induced differentiation. It provides strong technical support for subsequent development of turbot reproductive stem cells, efficient preservation and utilization of germplasm resources, and acceleration of the breeding process of new varieties.

[0005] To achieve the above objectives, the present invention is implemented through the following solution:

[0006] This invention provides a culture medium for in vitro induction of differentiation of turbot spermatogonial stem cells, the culture medium comprising the following components:

[0007] The basal culture medium consisted of L-15, 15% FBS, 1% fish serum (v / v), 1% embryonic protein (v / v, collected from fertilized turbot eggs about to hatch, washed multiple times, then homogenized with L-5 culture medium using a homogenizer, the homogenate was repeatedly freeze-thawed 3 times, centrifuged at 13000 rpm, 4℃ for 30 min, the supernatant was discarded, diluted with L-15 at a 1:1 ratio, then filtered sterilely through a 0.22 μm filter, the protein concentration was measured, and the final volume was adjusted to 18 g / L), bFGF 2 ng / ml, LIF 2 ng / ml, GDNF 40 ng / ml, SCF 100 ng / ml, EGF 10 ng / ml, 11-KT 10~100 ng / ml, DHP 10 ng / ml, HCG 1 IU / ml, RA 1.5 ng / ml, and melatonin 2.3 μg / ml.

[0008] This invention also provides a method for the in vitro directed differentiation of spermatogonial stem cells from long-term passage of turbot, the method comprising the following steps:

[0009] After the spermatogonial stem cells from long-term passage of turbot have been cultured to the point of confluence with the culture flask, the culture medium is replaced with the above-mentioned culture medium, and half of the culture medium is replaced every 3 to 4 days at 23°C.

[0010] Sperm were collected from the culture medium 10-21 days after induction of differentiation.

[0011] In this invention, the age of the turbot is ≤1 year;

[0012] In the actual operation of this invention, the spermatogonial stem cells are turbot spermatogonial stem cells that have undergone long-term passage culture, and the preparation method is based on the existing technology (CN114717184A) to establish long-term passage turbot spermatogonial stem cells.

[0013] In this invention, the method for collecting sperm is as follows:

[0014] (1) Collect the suspended sperm in the culture medium by collecting 5 ml of culture medium into a 15 ml centrifuge tube;

[0015] (2) Collect adherent sperm from the culture medium, add 1 ml of 0.05% trypsin to the culture flask, shake 3-4 times in an “∞” pattern to obtain some adherent sperm, neutralize the trypsin with L-15 medium containing 15% FBS to obtain a suspension containing the remaining adherent sperm.

[0016] (3) Combine the suspended sperm and the suspension containing the remaining adherent sperm to obtain a complete sperm suspension;

[0017] (4) Centrifuge the intact sperm suspension at 1500 rpm, take the precipitate and discard the supernatant, add passage culture medium to the precipitate (the preparation method is based on the existing technology CN114717184A) to obtain sperm to be artificially inseminated.

[0018] In the actual operation of this invention, after obtaining sperm, the above-mentioned culture medium is added to the cell culture flask containing sperm to continue inducing differentiation, and the first batch of induced differentiated sperm is used for artificial insemination first, and other batches are used second.

[0019] The present invention also provides a method for artificial insemination of turbot, the method comprising the following steps:

[0020] Collect 0.5 ml of well-developed female fish eggs and transfer them to a culture dish containing sperm for artificial insemination. Mix slowly and add 4 ml of seawater to the culture dish to activate the sperm. Artificial insemination of the turbot is completed after 1 hour.

[0021] This invention provides a turbot embryo, which is obtained by the above-described artificial insemination method.

[0022] This invention provides the above-mentioned culture medium, the above-mentioned in vitro directed differentiation method, the above-mentioned artificial insemination method, and the application of the above-mentioned turbot embryos in the preservation of turbot germplasm resources.

[0023] This invention provides the above-mentioned culture medium, the above-mentioned in vitro directed differentiation method, the above-mentioned artificial insemination method, and the application of the above-mentioned turbot embryos in accelerating the turbot breeding cycle.

[0024] This invention provides the above-mentioned culture medium, the above-mentioned in vitro directed differentiation method, the above-mentioned artificial insemination method, and the application of the above-mentioned turbot embryo in the study of turbot reproductive development mechanism.

[0025] Existing technologies have induced in vitro differentiation of spermatogonial stem cells from long-term passaged fish such as medaka, grouper, chub, redfin pufferfish, American shad, and anchovy, but none have yielded viable offspring. This invention addresses key bottlenecks in the in vitro induction of differentiation of long-term passaged spermatogonial stem cells, the collection of induced differentiated sperm, and artificial insemination. This invention optimizes parameters related to the composition of the differentiation induction culture medium, growth factors promoting cell proliferation and differentiation, the ratio of hormones inducing directional differentiation, the culture conditions for differentiation induction, sperm collection, and artificial insemination methods. This invention provides a method for the in vitro induction, collection, and artificial insemination of long-term passaged spermatogonial stem cells from turbot, enabling the production of viable offspring. Attached Figure Description

[0026] Figure 1The expression of rec8 under different concentrations of 11-KT induced differentiation was analyzed. Figure A shows the relative expression level of rec8 mRNA. In Figure A, groups B and C on the horizontal axis represent the groups before differentiation induction. The numbers 7, 14, and 21 on the horizontal axis of Figure A represent the 7th, 14th, and 21st days of differentiation induction, respectively. Figure B shows the Western blot of Rec8 protein.

[0027] Figure 2 This diagram illustrates the in vitro differentiation of spermatogonial stem cells induced by hormones. Figure A shows the state of spermatogonial stem cells before induction; Figure B shows a cell cluster 40 days after induction, composed of Sertoli cells and spermatogenic cells at various stages; Figure C is a magnified view of the cell cluster 40 days after induction; Figure D shows sperm production from a portion of the cell cluster 46 days after induction; Figure E shows the honeycomb structure formed by Sertoli cells after sperm detachment; Figure F is a magnified view of the honeycomb structure in Figure E; Figure G shows sperm activated by seawater after in vitro induction. Arrows in the figures point to sperm; the scale bar for Figures A-F is 50 μm, and the scale bar for Figure G is 20 μm.

[0028] Figure 3 GFP transgenic spermatogonial stem cells prepared by lentivirus transfection. Figures A and B show the fluorescence observations of spermatogonial stem cells and wild-type spermatogonial stem cells after six rounds of puromycin selection, respectively.

[0029] Figure 4 This section describes the in vitro differentiation of GFP transgenic spermatogonial stem cells. Figures A-C show GFP transgenic spermatogonial stem cells before induced differentiation, where A is the result under white light, B is the result under fluorescence, and C is a superimposed image of two lights. Figures D-F show the emergence of spermatogenic cells carrying GFP at various stages after 14 days of induced differentiation, where D is the result under white light, E is the result under fluorescence, and F is a superimposed image of two lights. Figures G-I show activated sperm carrying the GFP gene, where G is the result under white light, H is the fluorescence result when sperm motility is slow, and I is the fluorescence result when sperm motility is fast. Sperm circled in dashed lines in each figure are sperm carrying GFP green fluorescent protein; the scale bar for each figure is 50 μm.

[0030] Figure 5 In vitro differentiation of spermatogonial stem cells from different batches was performed. Figure A shows the in vitro differentiation of spermatogonial stem cells from juvenile fish. Figure B shows the differentiation of 1-year-old spermatogonial stem cells. + In vitro differentiation of spermatogonial stem cells from male fish after cryopreservation and thawing. Figure C shows 1-year-old spermatogonial stem cells. + In vitro differentiation of high-generation spermatogonial stem cells derived from male fish. Figure D shows the expression of differentiation genes during the differentiation process of spermatogonial stem cells from different groups. In Figure D, 1-3 on the horizontal axis represent spermatogonial stem cells derived from juvenile fish, spermatogonial stem cells after cryopreservation and thawing from adult fish, and high-generation spermatogonial stem cells derived from adult fish, respectively. The scale bar for each figure is 50 μm.

[0031] Figure 6This section describes the assessment of sperm quality and offspring acquisition through in vitro induced differentiation. Figure A shows sperm produced by induced differentiation (scale bar: 50 μm). Figure B shows ploidy detection by flow cytometry; in Figure B, BCs are diploid fish blood cells, IDSCs are sperm cells collected after induced differentiation, and ADCs are all cells collected from the culture flask after induced differentiation. Figure C shows routine seawater-activated sperm and scanning electron microscopy observation; in Figure C, a is activated normal sperm, b is abnormal sperm, c is normal sperm observed under electron microscopy, and d is abnormal sperm and cell debris (scale bar: 20 μm). Figure D shows fertilized eggs during embryogenesis and newly hatched larvae (scale bar: 500 μm).

[0032] Figure 7 This diagram illustrates cell differentiation caused by nutrient deficiency. Figure A shows cell aggregation after passage cessation; the solid red ellipses represent newly formed cell clusters, while the dashed white ellipses indicate further enlargement and differentiation. Figure B shows cells continuously producing sperm; the areas circled by the white curves represent differentiated sperm cells, white arrows indicate the locations of shed sperm, and red arrows indicate the locations of senescent cells. Figure C shows sperm motility after seawater activation. The scale bar for Figures A and B is 50 μm, and the scale bar for Figure C is 20 μm.

[0033] Figure 8 Immunofluorescence analysis of cells differentiated in vitro due to nutrient deprivation. Figure A shows DAPI staining of in vitro differentiated spermatogenic cell clusters. Figure B shows Vasa protein expression in in vitro differentiated spermatogenic cell clusters. Figure C shows an overlay of DAPI and Vasa in in vitro differentiated spermatogenic cell clusters; the area circled by the curve represents spermatogonial stem cell clusters. Figures D-F are magnified views of the boxes in Figures A-C, respectively. Arrows indicate sperm-like cells; the scale bar is 50 μm. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] Example 1

[0036] To screen for the optimal working concentration of 11-KT, three concentration gradients of 10 ng / ml, 100 ng / ml, and 1000 ng / ml were set up to conduct in vitro differentiation induction experiments on long-passaged spermatogonial stem cells. At the same concentration gradient, before 7 days of induced differentiation, the expression level of rec8b mRNA was not significantly different from that before induced differentiation (P > 0.05, P > 0.05, P > 0.05). At 14 days of induced differentiation, the relative expression level of rec8b mRNA in the 1000 ng / ml 11-KT concentration group was significantly upregulated compared to before and at 7 days of induced differentiation (P < 0.01, P < 0.001). At 21 days of induced differentiation, the relative expression levels of rec8b mRNA in the 10 ng / ml and 100 ng / ml concentration groups also began to show varying degrees of significant upregulation compared to the previous concentration groups. The differences among the three concentration groups were not significant, but the relative expression level of rec8b in the 10 ng / ml concentration group was higher than that in the other two high-concentration groups. Figure 1 A), and the 1000 ng / ml concentration group showed severe apoptosis.

[0037] Western blot results showed that Rec8 protein expression levels were upregulated on days 7, 14, and 21 after induced differentiation in different 11-KT concentration groups compared to pre-differentiation levels, but the differences were not significant during the induced differentiation period. In conclusion, a 10–100 ng / ml concentration of 11-KT is optimal for inducing differentiation.

[0038] Based on the concentrations selected from the 11-KT screening above, cells were seeded in six-well plates, and differentiation was induced once the cells reached confluence. Figure 2 A). As cells proliferate, cell clusters gradually form, consisting of supporting cells and spermatogenic cells. After 40 days of induced differentiation, these cell clusters appear in large numbers. Figure 2 B), and the differentiation phenomenon is obvious, with a large number of sperm cells or unactivated sperm visible around the cell cluster (B). Figure 2 C, D). Sperm collection during the peak differentiation period followed by seawater addition revealed motile sperm, with activity lasting between 3 and 29 minutes. In vitro differentiation in turbot, similar to in vivo spermatogenesis, involves batch production of sperm, from the initial discovery of differentiated sperm (8 days of induction) to the telogen stage, approximately 3 months. After the maturing sperm detach from the bottom of the culture plate, the remaining supporting cells form a honeycomb structure, similar to the spermatids without spermatogenic cells in the testes of fish. Figure 2 E, F).

[0039] Example 2

[0040] To more intuitively observe the differentiation process of spermatogonial stem cells, spermatogonial stem cells were transduced with the GFP (green fluorescent protein) gene.

[0041] 48 hours after eGFP+PURO Lentivirus transfection of spermatogonial stem cells, fluorescence observation revealed cells expressing GFP. Figure 3 A, Figure 3 B represents fluorescence observation of wild-type spermatogonial stem cells. 100 cells were counted, of which approximately 30% were GFP-positive. After screening with puromycin 6 times, more than 70% of the cells were positive.

[0042] As the number of passages increases, the proportion of positive cells decreases, but a certain proportion of positive cells carrying the GFP gene can still be observed. Figure 4 (A~C). Once the cells have filled the entire flask, the cells are predominantly spermatogonial stem cells, which aggregate with supporting cells to form flower-like cell clusters. Figure 4 A, C). 14 days after adding induction differentiation medium ( Figure 4 (D~F) The original flower-like cell clusters continued to aggregate to form multi-layered cell clusters, with sperm cells of 2~3 μm in diameter appearing above the cell clusters. After collecting and activating sperm, it was found that the activated sperm contained both GFP transgenes and sperm without GFP transgenes. When sperm motility was weak, the sperm head expressed strong green fluorescent protein, and when the sperm moved rapidly, obvious green trails could be seen. Figure 4 (G~I) further proved that spermatogonial stem cells stably cultured in vitro, after being artificially supplemented with various hormones to simulate the in vivo spermatogenesis environment, can produce viable sperm.

[0043] Example 3

[0044] To further investigate whether this method is applicable to inducing differentiation of different spermatogonial stem cell lines, and which cell line is more prone to differentiation, spermatogonial stem cells derived from juvenile fish (referred to as Group A) and 1-year-old spermatogonial stem cells were compared. + Spermatogonial stem cells from male fish that had been cryopreserved for 16 months (referred to as Group B) and spermatogonial stem cell lines that had been stably passaged to 130 generations (referred to as Group C) were subjected to hormone-induced differentiation.

[0045] The results showed that, under the same culture conditions, after 4 days of passage culture, the spermatogonial stem cells in group C exhibited the most vigorous growth. Figure 5 (The cells) resembled pebbles. At 34 days, all three spermatogonial stem cell lines showed obvious cell clusters, which were composed of spermatogenic cells and supporting cells. In groups A and B, the supporting cells at the bottom layer of the spermatogonial stem cell clusters were clearly visible, resembling solar radiation, and spermatocytes had differentiated around the periphery of the cell clusters. In group C, in addition to cell clusters, spermatocytes and spermatocytes of the same differentiation level, resembling bunches of grapes, appeared on the supporting cells.

[0046] Sperm were collected and activated, and all three cell lines produced motile sperm. The spermatogonial stem cell line in group A produced fewer sperm than the other two cell lines. Figure 5 (A~C) , and at 50 days of induced differentiation, most cell clusters consisted only of supporting cells, with a significant reduction or even disappearance of spermatogenic cells. At 50 days of induced differentiation, the spermatogonial stem cell lines in groups B and C exhibited a state similar to seminiferous vesicle cells, containing spermatogenic cells at different stages. Spermatoglyphics were present near epithelial-like tissue, and spermatocytes or spermatocytes at the same stage clustered together in a grape-like morphology. Sperm collection and activation of cells around 50 days of induced differentiation revealed that while group A had sperm, they failed to activate. The other two cell lines had viable sperm, albeit in reduced numbers. At 80 days of induced differentiation, sperm collection and activation showed no further viable sperm production. The cell clusters established in group A gradually disappeared, and a monolayer of spermatogonial stem cells reappeared. The other two cell lines showed many vacuolated cell clusters or senescent cells, and in some areas, the pre-differentiation cell state reappeared. Simultaneously, at 21 days of induced differentiation, the expression of sysp3, dmc1, and rec8 was detected in both groups. Figure 5 D). In summary, 1-year-old is preferred. + Stable passage of spermatogonial stem cells.

[0047] Example 4

[0048] Following the optimal protocol, 42 generations of Itga10-overexpressing spermatogonia and over 300 generations of spermatogonia were induced to differentiate for about half a month, resulting in cell clusters that had differentiated into sperm cells around their periphery. Figure 6 A), after slowly digesting and collecting the culture medium with 0.05% trypsin, and using normal diploid fish blood as a control, flow cytometry analysis showed that the ploidy of the digested cells was mainly in haploid form. Figure 6 B), simultaneous detection of ploidy in all cells in the entire culture flask revealed haploid peaks and aneuploid peaks larger than diploid peaks.

[0049] In vitro differentiated sperm cells were activated and tested with seawater. The results showed the presence of sperm with long flagella that swim rapidly, as well as abnormal sperm. Scanning electron microscopy further confirmed the presence of sperm with normal morphology and abnormal sperm with only a head without flagella or with short flagella. Figure 6 C).

[0050] The collected sperm was used for artificial insemination with normal female fish eggs, with male fish semen as a control. The fertilization rate and hatching rate were statistically analyzed (Table 1). Finally, 9 normally developed fry were obtained. Figure 6 (D) Although the number of viable cells obtained was small, it represents a breakthrough in inducing differentiation of long-term passaged spermatogonial stem cells. To date, there have been no reports of sperm produced from the induction of long-term passaged spermatogonial stem cells possessing fertilization and hatching capabilities.

[0051] Table 1 Fertilization rate and hatching rate

[0052] No source sperm Number of eggs Fertilization rate (%) Hatching rate (%) live sperm 828 95.0% 100% Itga10 overexpression induces spermatogenesis in spermatogonia. 619 1.13 100% Sperm differentiation from spermatogonia of 300 generations or more in vitro 480 0.42 100%

[0053] Comparative Example 1

[0054] Compared with Examples 1 and 4, which involved inducing differentiation for more than half a month, the control example used a nutrient deficiency method and did not add hormones.

[0055] After passage was stopped, cell changes were observed. Once sperm cells appeared, the cells were collected by trypsin digestion, washed twice with L-15 culture medium containing 10% FBS, and finally brought to a final volume of 1.5 ml. One drop was taken with a pipette and placed on a glass slide, and a small amount of seawater was added to stimulate observation to see if there were any motile sperm.

[0056] After passage has been stopped for more than half a month, spermatogenic cells and supporting cells gradually form cell clusters. Once these clusters grow to a certain size, they form multi-layered cell masses. Spermatogonia further differentiate into sperm-like cells. Figure 7 A, B). Vasa immunofluorescence analysis of the differentiated cells revealed that spermatogonial stem cells and spermatocytes expressed Vasa protein (red light), while sperm cells did not express Vasa protein. This further demonstrated that spermatogonial stem cells differentiated into 2-3 μm spermatids, with the spermatids mainly distributed on the periphery of the cell cluster. Spermatogonia and spermatocytes (expressing Vasa, red light) were distributed below the cell cluster, closely adjacent to the supporting cells, and adhered to the bottom of the culture plate. Figure 8 After collecting and activating the sperm, motile sperm can be seen. Figure 7 C), the number of sperm was much smaller than that produced by hormone induction in Examples 1 and 4, and the number of abnormal sperm was also greater than that in Examples 1 and 4 at the same induction differentiation stage. Figure 6 C), and the differentiation period is very short. In the induced differentiated cells, vacuolated structures appear much faster than in the previous example due to the shedding of mature sperm and senescent cells (see [example]). Figure 7 B).

[0057] Comparative Example 2

[0058] Based on the optimal ratio provided in the example, 1000 ng / ml 11-KT was added to induce differentiation. At 14 days, the relative expression level of rec8b mRNA was significantly upregulated (P < 0.01, P < 0.001), but the protein level did not change, and apoptosis was obvious, which was not conducive to subsequent sperm collection.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A culture medium for in vitro induction of differentiation of turbot spermatogonial stem cells, characterized in that, The culture medium consists of the following components: The basal culture medium consisted of L-15, 15% FBS, 1% fish serum, 1% embryonic protein extract, bFGF 2 ng / ml, LIF 2 ng / ml, GDNF 40 ng / ml, SCF 100 ng / ml, EGF 10 ng / ml, 11-KT 10~100 ng / ml, DHP 10 ng / ml, HCG 1 IU / ml, RA 1.5 ng / ml, and melatonin 2.3 μg / ml.

2. A method for the in vitro directed differentiation of spermatogonial stem cells from turbot through long-term passage, characterized in that, The in vitro directed differentiation method includes the following steps: After the spermatogonial stem cells of turbot have been passaged for a long time and have been cultured to the full extent of the culture flask, the culture medium is replaced with the culture medium described in claim 1, and half of the culture medium is replaced every 3 to 4 days at 23°C. Sperm were collected from the culture medium 10-21 days after induction of differentiation.

3. The in vitro directed differentiation method according to claim 2, characterized in that, The age of the turbot is ≤1 year.

4. The in vitro directed differentiation method according to claim 2, characterized in that, The method for collecting the sperm is as follows: (1) Collect the suspended sperm in the culture medium by collecting 5 ml of culture medium into a 15 ml centrifuge tube; (2) Collect adherent sperm from the culture medium, add 1 ml of 0.05% trypsin to the culture flask, shake 3-4 times to obtain some adherent sperm, neutralize the trypsin with L-15 medium containing 15% FBS to obtain a suspension containing the remaining adherent sperm. (3) Combine the suspended sperm and the suspension containing the remaining adherent sperm to obtain a complete sperm suspension; (4) Centrifuge the intact sperm suspension at 1500 rpm, take the precipitate and discard the supernatant, add passage culture medium to the precipitate to obtain sperm for artificial insemination.

5. A method for artificial insemination of turbot, characterized in that, The method includes the following steps: Collect 0.5 ml of well-developed female fish eggs and transfer them to a culture dish containing sperm for artificial insemination. Add 4 ml of seawater to the culture dish to activate the sperm. After 1 hour, artificial insemination of the turbot is completed.

6. A turbot embryo, characterized in that, The embryo was obtained by the artificial insemination method described in claim 5.

7. The culture medium according to claim 1, the in vitro directed differentiation method according to any one of claims 2 to 4, the artificial insemination method according to claim 5, and the application of turbot embryos according to claim 6 in the preservation of turbot germplasm resources.

8. The culture medium of claim 1, the in vitro directed differentiation method of any one of claims 2 to 4, the artificial insemination method of claim 5, and the application of the turbot embryo of claim 6 in accelerating the turbot breeding cycle.

9. The application of the culture medium according to claim 1, the in vitro directed differentiation method according to any one of claims 2 to 4, the artificial insemination method according to claim 5, and the turbot embryo according to claim 6 in the study of turbot reproductive development mechanism.

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