A method for evaluating the effect of hormone-induced ovarian development in Japanese eel

Abstract

The present invention discloses a method for evaluating the effect of hormone-induced ovarian development of Japanese eels. The method comprises the following steps: S1: selecting female Japanese eels, providing non-denatured mammalian protein extract, FITC, and hCG. S2: obtaining serum containing vitellogenin. S3: obtaining purified vitellogenin. S4: coupling labeling treatment, verifying the purification effect. S5: setting up experiments to verify whether the target hormone can promote vitellogenin to enter oocytes, thereby effectively inducing ovarian development of Japanese eels. S6: observing the fluorescence intensity of the experimental group under a fluorescence microscope, verifying whether the target hormone can promote vitellogenin to enter oocytes based on Western Blot, and evaluating the induced development effect. The present invention is based on judging whether the target hormone can promote vitellogenin to enter oocytes, thereby effectively inducing ovarian development of Japanese eels, and is of great significance for accurately evaluating the effect of a new ripening agent on inducing yolk accumulation, thereby providing technical support for studying artificial breeding of Japanese eels.

Description

Technical Field

[0001] The invention relates to the technical field of fish physiology research, and in particular to a method for evaluating the effect of hormone-induced ovarian development of Japanese eel. Background Art

[0002] Japanese eel (Anguilla japonica) is one of the most important migratory fish in Asia and an important economic fish in my country. Due to the particularity of the life history of Japanese eel, its artificial breeding technology is a global problem that has not yet been solved. The seedlings required for artificial breeding have always relied on natural fishing. After the 1980s, with the rapid development of eel farming, my country has become one of the countries with the highest degree of industrialization of eel farming in the world. However, since all the seedlings required for farming rely on natural fishing, this predatory farming method has caused the natural eel seed resources to decline year by year. The shortage of eel seedlings has restricted the further development of the eel industry. Therefore, research on artificial breeding technology of eels is particularly important for protecting natural resources and the sustainable development of the eel farming industry.

[0003] Artificial breeding of eels began in the 1930s. In 1934, Boucher et al. first artificially induced the testes of male European eels to mature. In 1964, Fontaine et al. induced female European eels to ovulate. In 1978, Epler et al. used the same method to induce European eels to spawn and obtained early embryos. In the 1960s, Japanese scholars began research on artificial breeding of Japanese eels. In 1970, Ishida et al. first induced the ovaries of Japanese eels to mature and ovulate. In 1972, Yamamoto et al. obtained the first batch of young eels, although they died after surviving for 120 days, which was the first time in the world. It was not until 1999 that Tanaka et al. used shark egg freeze-dried powder as the main ingredient of Japanese eel breeding feed, making a major breakthrough, allowing artificially hatched young eels to survive for 253 days. In 2003, the Japan Fisheries Research Institute reported again that the survival time of artificially hatched eels reached 612 days, and the maximum individual body length exceeded 20cm. In March 2010, the Fisheries Research Center under the Japan Fisheries Agency injected hormones into 50 eels to ripen them, and obtained 250,000 fertilized eggs. By late April, only 10,000 fry survived. Although the artificial breeding technology of eels has made significant progress, it is still far from the demand for industrial large-scale breeding and production of eel fry. The main reason is that the maturity rate, spawning rate and egg production of artificially ripened eels are still relatively low, especially the quality of the eggs produced is not ideal, resulting in low fertilization rate, hatching rate and survival rate. Although the hatched eels can eventually grow into white eel fry after metamorphosis, the growth rate is very slow, the survival rate is extremely low, and the deformity rate is high. In addition to the above shortcomings, the cost of artificially cultivated starter bait, breeding and cultivation is as high as thousands of dollars per eel. Such high costs are a fatal limitation to industrialization. The key to solving these problems lies in how to improve the quality of eggs of artificially bred eels, which requires systematic and in-depth research and experiments.

[0004] In order to explore solutions to improve egg quality, it is first necessary to establish evaluation standards for high-quality and healthy eggs. However, the current evaluation methods for fish egg quality are mostly limited to the observation of phenotypic data of egg cells (such as diameter measurement), while the evaluation methods for their internal nutrients, especially the amount of vitellogenin (VTG), the most important nutrient for embryonic and post-embryonic development, entering the eggs is still blank. On the other hand, as the most important stage in the process of oogenesis, yolk production plays a decisive role in the quality of mature eggs, and real-time monitoring of this stage can more intuitively judge the specific state of oogenesis. Therefore, the development of detection technology for yolk protein intake can be applied to the evaluation of egg quality, especially for accurately evaluating the effect of new ripening agents in inducing yolk accumulation. Summary of the invention

[0005] Based on this, it is necessary to provide an evaluation method for the effect of hormone-induced ovarian development in Japanese eels to address the current gap in yolk protein intake detection technology.

[0006] A method for evaluating the effect of hormone-induced ovarian development in Japanese eel, comprising the following steps:

[0007] S1 selected female Japanese eels and provided estradiol, 1× PBS buffer, non-denaturing mammalian protein extract, and FITC;

[0008] S2 first injecting the female Japanese eel with the estradiol to obtain serum containing vitellogenin;

[0009] S3: the serum containing vitellogenin is purified by the 1×PBS buffer and the non-denaturing mammalian protein extract to obtain purified vitellogenin;

[0010] S4 uses the FITC to couple and label the purified vitellogenin, and verifies the purification effect based on SDS-PAGE electrophoresis;

[0011] S5 set up experiments to verify whether the target hormone can effectively induce the ovarian development of Japanese eel;

[0012] The specific operation of step S5 is as follows:

[0013] S51 taking the ovarian tissue of the female Japanese eel, placing it in a 24-well cell culture plate after treatment, and adding culture medium for culturing;

[0014] S52 sets up a control experiment, and chooses whether to add the target hormone and FITC-labeled vitellogenin to the cell culture plate according to the experimental requirements;

[0015] The control experiment was set up as follows:

[0016] Experimental group 1: addition of vitellogenin without FITC labeling;

[0017] Experimental group 2: addition of target hormone and vitellogenin without FITC labeling;

[0018] Experimental group 3: addition of FITC-labeled vitellogenin;

[0019] Experimental group 4: adding target hormone and FTTC-labeled vitellogenin;

[0020] Experimental group 5: no target hormone and FITC-labeled vitellogenin were added;

[0021] Set three repetitions per set;

[0022] S6 Observe the fluorescence intensity of the experimental group under a fluorescence microscope, verify based on Western Blot whether the target hormone can promote the entry of vitellogenin into oocytes, and evaluate the induced development effect.

[0023] The above evaluation method is based on the model that Japanese eel oocytes are cultured in a mixed culture medium containing the target hormone (hCG) and FITC-coupled labeled vitellogenin, combined with fluorescence microscopy observation and Western Blot verification. It is used to determine whether the target hormone (hCG) can promote the entry of vitellogenin into oocytes, thereby effectively inducing ovarian development in Japanese eels. This is of great significance for accurately evaluating the effect of new ripening agents in inducing yolk accumulation, thereby providing technical support for the study of artificial breeding of Japanese eels.

[0024] In one embodiment, the selection criteria of the female Japanese eel are as follows: strong physique, body weight of 1±0.2 kg; the non-denatured mammalian protein extract has the following components: 50 mM NaH 2 PO 4 , 300mM NaCl, 0.05% Tween 20.

[0025] Furthermore, the pH value of the non-denatured mammalian protein extract is adjusted to 8.0 using NaOH.

[0026] In one embodiment, the specific operation of step S2 is as follows:

[0027] S21 injecting the estradiol into the female Japanese eel;

[0028] S22 extracts blood from the Japanese eel, centrifuges it and then absorbs the serum containing vitellogenin.

[0029] Furthermore, the injection standard of estradiol is: once every two days, the injection dose is 10 mg / kg, and the injection is continued for two weeks.

[0030] In one embodiment, the purification method is specifically performed as follows:

[0031] S31 provides ultrafiltration tubes and sterilized ultrapure water;

[0032] S32: Fill the ultrafiltration tube with the sterilized ultrapure water, precool it on ice for 10 minutes, and discard the ultrapure water;

[0033] S33 was added with 1× PBS buffer, centrifuged and the filtrate was discarded;

[0034] S34 adding the serum containing vitellogenin, centrifuging and discarding the filtrate;

[0035] S34: continue to add the non-denatured mammalian protein extract, centrifuge for multiple times, discard the filtrate, and obtain purified vitellogenin.

[0036] Furthermore, after purification, most of the impurities were filtered out through the filter membrane, and then SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining were performed to observe the protein bands.

[0037] In one embodiment, the specific operation of step S4 is as follows:

[0038] S41 provides Na 2 CO 3 / NaHCO 3 Buffer, 1× PBS buffer;

[0039] S42 uses the Na 2 CO 3 / NaHCO 3 After dissolving the FITC in a buffer solution, the buffer solution is mixed with the purified vitellogenin to obtain a coupling solution;

[0040] S43: Add the coupling liquid into the ultrafiltration tube, add the Na 2 CO 3 / NaHCO 3 The coupling solution is washed by centrifugation with a buffer until there is no yellow-green color in the filtrate;

[0041] S44 Continue to add 1× PBS buffer to the ultrafiltration tube and centrifuge. Repeat 3 times to remove Na 2 CO 3 / NaHCO 3 The buffer was replaced with 1× PBS buffer, and SDS-PAGE electrophoresis was performed to verify the coupling labeling effect.

[0042] Furthermore, the Na 2 CO 3 / NaHCO 3 The concentration of the buffer was 0.05 M and the pH was 9.5.

[0043] In one embodiment, the culture medium has the following components: 1% tertiary antibody, 10% fetal bovine serum, and L-15 basal culture medium; wherein the tertiary antibody is obtained by mixing 10000 units / ml penicillin, 10 mg / ml streptomycin, and 1250 units / ml nystatin.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The evaluation method of the present invention is based on a mode in which Japanese eel oocytes are cultured in a mixed culture medium containing a target hormone (hCG) and FITC-labeled vitellogenin, and combined with fluorescence microscope observation and Western Blot verification, to judge whether the target hormone (hCG) can promote vitellogenin to enter the oocyte, thereby effectively inducing ovarian development of the Japanese eel. This method is of great significance for accurately evaluating the effect of a new ripening agent on inducing yolk accumulation, thereby providing technical support for studying artificial reproduction of Japanese eels. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Shown is the SDS-PAGE gel electrophoresis diagram for verifying the purification effect of vitellogenin.

[0047] Figure 2 Shown is the result of SDS-PAGE gel electrophoresis obtained under blue light excitation to verify the effect of vitellogenin fluorescence labeling.

[0048] Figure 3 Shown are the experimental results of Japanese eel ovarian tissue culture under fluorescence microscope observation.

[0049] Figure 4 Shown are the results of Western Blot experiments to verify the effect of hCG in inducing ovarian development in Japanese eel. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings.

[0051] The present invention provides a method for evaluating the effect of hormone-induced ovarian development in Japanese eel, which comprises the following steps:

[0052] S1 selected female Japanese eels, provided estradiol, 1× PBS buffer, non-denaturing mammalian protein extract, and FITC (fluorescein isothiocyanate).

[0053] The selection criteria for female Japanese eels are as follows: strong physique and body weight of 1±0.2 kg.

[0054] Non-denaturing mammalian protein extract has the following components: 50mM NaH 2 PO 4 , 300 mM NaCl, 0.05% Tween 20 (non-ionic surfactant), wherein the pH value of the non-denatured mammalian protein extract was adjusted to 8.0 with NaOH.

[0055] S2: injecting the estradiol into the female Japanese eel to obtain serum containing vitellogenin.

[0056] The specific operation of step S2 is as follows:

[0057] S21: injecting estradiol into the female Japanese eel.

[0058] The injection standard of estradiol is: once every two days, the injection dose is 10mg / kg, and the injection is continuous for two weeks.

[0059] S22 extracts blood from the female Japanese eel, centrifuges the blood and then extracts serum containing vitellogenin.

[0060] The serum containing vitellogenin S3 was purified by 1×PBS buffer and non-denaturing mammalian protein extract to obtain purified vitellogenin.

[0061] The specific operation of the purification method is as follows:

[0062] S31 provides ultrafiltration tubes and sterilized ultrapure water.

[0063] S32 Fill the ultrafiltration tube with sterile ultrapure water, pre-cool it on ice for 10 minutes, and pour out the ultrapure water.

[0064] S33 was added with 1× PBS buffer, and the filtrate was discarded after centrifugation.

[0065] S34 was added with serum containing vitellogenin, and the filtrate was discarded after centrifugation.

[0066] S34 continues to add non-denatured mammalian protein extract, centrifuges for multiple times, discards the filtrate, and obtains purified vitellogenin.

[0067] S4 used FITC to couple the purified vitellogenin and verified the purification effect based on SDS-PAGE electrophoresis.

[0068] The specific operation of step S4 is as follows:

[0069] S41 provides Na 2 CO 3 / NaHCO 3 Buffer, 1× PBS buffer.

[0070] Na 2 CO 3 / NaHCO 3 The concentration of the buffer was 0.05 M and the pH was 9.5.

[0071] S42 uses Na 2 CO 3 / NaHCO 3 After FITC is dissolved in the buffer, it is mixed with purified vitellogenin to obtain a coupling solution.

[0072] S43: Add the coupling liquid into the ultrafiltration tube, add the Na 2 CO 3 / NaHCO 3 The coupling solution is washed by centrifugation with a buffer until there is no yellow-green color in the filtrate;

[0073] S44 Continue to add 1× PBS buffer to the ultrafiltration tube and centrifuge. Repeat 3 times to remove Na 2 CO 3 / NaHCO 3 The buffer was replaced with 1× PBS buffer, and SDS-PAGE electrophoresis was performed to verify the coupling labeling effect.

[0074] S5 set up experiments to verify whether the target hormone (hCG) can effectively induce ovarian development in Japanese eel.

[0075] The specific operation of step S5 is as follows:

[0076] S51 Ovarian tissue of female Japanese eel was obtained, processed and placed in a 24-well cell culture plate, and cultured with culture medium.

[0077] The culture medium has the following components: 1% tertiary antibody, 10% fetal bovine serum, and L-15 basal culture medium, wherein the tertiary antibody is obtained by mixing 10000 units / ml penicillin, 10 mg / ml streptomycin, and 1250 units / ml nystatin.

[0078] S52 sets up a control experiment and chooses whether to add the target hormone (hCG) and FITC-labeled vitellogenin to the cell culture plate according to the experimental requirements.

[0079] The control experiment was set up as follows:

[0080] Experimental group 1: Addition of vitellogenin without FITC labeling.

[0081] Experimental group 2: hCG and vitellogenin without FITC labeling were added.

[0082] Experimental group 3: FITC-labeled vitellogenin was added.

[0083] Experimental group 4: hCG and FTTC-labeled vitellogenin were added.

[0084] Experimental group 5: no hCG and FITC-labeled vitellogenin were added.

[0085] Set up three repetitions per set.

[0086] S6 observed the fluorescence intensity of the experimental group under a fluorescence microscope, verified whether hCG could promote the entry of vitellogenin into oocytes based on Western Blot, and evaluated the induced development effect.

[0087] In summary, the present invention cultured Japanese eel oocytes in a mixed culture medium containing a target hormone (hCG) and FITC-labeled vitellogenin, and could judge whether the target hormone could promote the entry of vitellogenin into oocytes by means of fluorescence microscopy observation and Western Blot verification, thereby effectively inducing ovarian development of Japanese eels, which is of great significance for accurately evaluating the effect of a new ripening agent on inducing yolk accumulation, thereby providing technical support for studying artificial breeding of Japanese eels.

[0088] Example 1

[0089] Acquisition, purification and FITC-coupled labeling of vitellogenin from Japanese eel.

[0090] Obtaining vitellogenin: Select female Japanese eels with a strong physique and a body weight of 1±0.2 kg, and inject them with estradiol once every two days for two weeks. The injection dose is 10 mg / Kg. Draw blood into a centrifuge tube, let it stand in a 4°C refrigerator overnight, and then centrifuge it at 6000 rpm for 10 minutes. Aspirate the serum, and vitellogenin is present in the serum.

[0091] Purification of vitellogenin: Fill a 4ml ultrafiltration tube with sterile ultrapure water of specification 100kDa, pre-cool on ice for 10 minutes, pour out the ultrapure water, replace the sterile ultrapure water with 1×PBS buffer, centrifuge at 4000rcf for 15 minutes after adding, discard the filtrate, continue to add serum, centrifuge at 4000rcf for 15 minutes, discard the filtrate, then add non-denatured mammalian protein extract, centrifuge at 4000rcf for 15 minutes, discard the filtrate, repeat 5 times. After purification, most of the impurities are filtered out through the filter membrane.

[0092] SDS-PAGE and Coomassie Brilliant Blue staining were performed to observe the protein bands. Figure 1 As shown, Figure 1 The figure shows the SDS-PAGE gel electrophoresis diagram for verifying the purification effect of vitellogenin, where:

[0093] M: protein molecular weight standard; 1: serum protein without estradiol injection; 2: serum protein with estradiol injection; 3: protein after purification for 3 times; 4: protein after purification for 5 times; 5: filtrate of the last purification. The black arrow indicates the protein band represented by vitellogenin. The above results show that this purification method can effectively purify vitellogenin in serum.

[0094] FITC coupling labeling: use a concentration of 0.05 M Na 2 CO 3 / NaHCO 3 Dissolve FITC in buffer, mix with purified vitellogenin, couple overnight in 4°C refrigerator, add to ultrafiltration tube, centrifuge at 4000rcf for 15 min, add Na 2 CO 3 / NaHCO 3 Buffer, centrifuge at 4000rcf for 15min to wash away excess FITC, repeat several times until there is no yellow-green color in the filtrate, add 1×PBS buffer to the ultrafiltration tube, centrifuge at 4000rcf for 15min, repeat 3 times, and remove Na 2 CO 3 / NaHCO 3 The buffer was replaced with 1× PBS buffer and the purification effect was verified by SDS-PAGE electrophoresis. The verification results were as follows: Figure 2 As shown, Figure 2 The results of SDS-PAGE gel electrophoresis under blue light excitation to verify the effect of vitellogenin fluorescence labeling are shown, where: 1: vitellogenin without FITC coupling labeling; 2: vitellogenin with FITC coupling labeling. The black arrow indicates the protein band represented by vitellogenin. The results show that this labeling method can label vitellogenin with FITC.

[0095] Example 2

[0096] The ovarian tissue culture and fluorescence microscopy observation of Japanese eel were used to examine the effect of hCG on inducing ovarian development of Japanese eel.

[0097] We selected healthy female Japanese eels, took out their ovarian tissue after anesthesia, cut them into small pieces with scissors, and added them to a 24-well cell culture plate. We set up a control experiment to explore whether hCG can effectively induce ovarian development in Japanese eels. The experiment was divided into five groups:

[0098] Experimental group ①: 100 μL of vitellogenin without FITC labeling and 100 μL of 1× PBS buffer were added;

[0099] Experimental group ②: 100 μL of vitellogenin without FITC labeling and 100 μL of hCG with a concentration of 1000 IU / ml were added;

[0100] Experimental group ③: 100 μL FITC-labeled vitellogenin and 100 μL 1× PBS buffer were added;

[0101] Experimental group ④: 100 μL FITC-labeled vitellogenin and 100 μL hCG at a concentration of 1000 IU / ml were added;

[0102] Experimental group ⑤: add 200 μL 1× PBS buffer;

[0103] Three replicates were set up for each group. 1 ml of complete culture medium (containing 1% triple antibody, 10% fetal bovine serum, and L-15 basal culture medium) was added to each well, and the 24-well cell culture plate was placed in a 37°C incubator. After two days, the liquid was aspirated, and 1× PBS buffer was added to rinse the ovarian tissue fragments. The cell culture plate was placed under a fluorescence microscope for observation. The results were as follows: Figure 3 shown. Figure 3 The results of the experiment of Japanese eel ovarian tissue culture under fluorescence microscope observation are shown. "+" indicates addition; "-" indicates no addition. The results show that hCG can promote the entry of vitellogenin into oocytes, thereby effectively inducing the development of Japanese eel ovaries.

[0104] Example 3

[0105] Western Blot was used to verify the effect of hCG on inducing the development of the ovary of Japanese eel.

[0106] The ovarian tissue of Japanese eel in the cell culture plate was collected, and the tissue protein was extracted by trizol method. The protein concentration was measured by BCA method and diluted to the same concentration. 80 μL of sample and 20 μL of 5× protein loading buffer were mixed, and the mixture was incubated at 95°C for 10 min. Western Blot was used to verify whether hCG could promote the entry of vitellogenin into oocytes (the primary antibody used was FITC antibody). Figure 4 As shown, Figure 4 The results of the Western Blot experiment verifying the effect of hCG on the ovarian development of Japanese eels. "VTG" represents vitellogenin after FITC coupling labeling. The primary antibody used in the experiment is FITC antibody. "+" means added; "-" means not added. The results show that hCG can promote the entry of vitellogenin into oocytes, thereby effectively inducing the ovarian development of Japanese eels.

Claims

1. A method for evaluating the effect of hormone-induced ovarian development in Japanese eel, characterized in that: The following steps are involved: S1 selected female Japanese eels and provided estradiol, 1× PBS buffer, non-denaturing mammalian protein extract, and FITC; S2 first injecting the female Japanese eel with the estradiol to obtain serum containing vitellogenin; S3: the serum containing vitellogenin is purified by the 1×PBS buffer and the non-denaturing mammalian protein extract to obtain purified vitellogenin; S4 uses the FITC to couple and label the purified vitellogenin, and verifies the purification effect based on SDS-PAGE electrophoresis; S5 set up experiments to verify whether the target hormone can effectively induce the ovarian development of Japanese eel; The specific operation of step S5 is as follows: S51 taking the ovarian tissue of the female Japanese eel, placing it in a 24-well cell culture plate after treatment, and adding culture medium for culturing; S52 sets up a control experiment, and chooses whether to add the target hormone and FITC-labeled vitellogenin to the cell culture plate according to the experimental requirements; The control experiment was set up as follows: Experimental group 1: addition of vitellogenin without FITC labeling; Experimental group 2: addition of target hormone and vitellogenin without FITC labeling; Experimental group 3: addition of FITC-labeled vitellogenin; Experimental group 4: adding target hormone and FITC-labeled vitellogenin; Experimental group 5: no target hormone and FITC-labeled vitellogenin were added; Set three repetitions per set; S6 Observe the fluorescence intensity of the experimental group under a fluorescence microscope, verify based on Western Blot whether the target hormone can promote the entry of vitellogenin into oocytes, and evaluate the induced development effect.

2. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 1, characterized in that: The selection criteria of the female Japanese eel are as follows: strong physique, body weight of 1±0.2 kg; The non-denaturing mammalian protein extract has the following components: 50 mM NaH2PO4, 300 mM NaCl, and 0.05% Tween 20.

3. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 2, characterized in that: The pH value of the non-denatured mammalian protein extract is adjusted to 8.0 with NaOH.

4. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 1, characterized in that: The specific operation of step S2 is as follows: S21 injecting the estradiol into the female Japanese eel; S22 extracts blood from the Japanese eel, centrifuges it and then absorbs the serum containing vitellogenin.

5. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 4, characterized in that: The injection standard of estradiol is: once every two days, the injection dose is 10 mg / kg, and the injection is continued for two weeks.

6. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 1, characterized in that: The purification method is specifically performed as follows: S31 provides ultrafiltration tubes and sterilized ultrapure water; S32: Fill the ultrafiltration tube with the sterilized ultrapure water, precool it on ice for 10 minutes, and discard the ultrapure water; S33 was added with 1× PBS buffer, centrifuged and the filtrate was discarded; S34 adding the serum containing vitellogenin, centrifuging and discarding the filtrate; S34: continue to add the non-denatured mammalian protein extract, centrifuge for multiple times, discard the filtrate, and obtain purified vitellogenin.

7. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 6, characterized in that: After purification, most of the impurities were filtered out through the filter membrane, and then SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining were performed to observe the protein bands.

8. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 1, characterized in that: The specific operation of step S4 is as follows: S41 provided Na2CO3 / NaHCO3 buffer and 1× PBS buffer; S42 uses the Na2CO3 / NaHCO3 buffer to dissolve the FITC, mixes it with the purified vitellogenin, and leaves it to stand at 4°C overnight to obtain a coupling solution; S43: adding the coupling liquid into the ultrafiltration tube, adding the Na2CO3 / NaHCO3 buffer solution for multiple times and centrifuging and washing the coupling liquid until there is no yellow-green color in the filtrate; S44 Continue to add 1×PBS buffer to the ultrafiltration tube and centrifuge, repeat 3 times, replace the Na2CO3 / NaHCO3 buffer with 1×PBS buffer, and perform SDS-PAGE electrophoresis to verify the coupling labeling effect.

9. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 8, characterized in that: The concentration of the Na2CO3 / NaHCO3 buffer is 0.05 M and the pH is 9.

5.

10. The method for evaluating the effect of hormone-induced ovarian development in Japanese eel according to claim 1, characterized in that: The culture medium has the following components: 1% triple antibody, 10% fetal bovine serum, and L-15 basic culture medium; The three antibodies were obtained by mixing 10000 units / ml penicillin, 10 mg / ml streptomycin and 1250 units / ml nystatin.

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