Novel fish oxytocic as well as use method and application thereof

Abstract

The invention discloses a novel fish oxytocic as well as a use method and application thereof, and belongs to the technical field of fish oxytocic. The novel fish oxytocic takes polypeptide as a main component or an effective component, and the amino acid sequence of the polypeptide is shown as SEQ ID NO.1. After intraperitoneal injection of the novel fish oxytocic provided by the invention, the novel fish oxytocic has a remarkable oxytocic effect on zebrafish and can be applied to ovulation promotion of artificially cultured fishes; the method has important significance in reducing the adverse effect of external adverse environment on fish ovulation, reducing the condition requirements in the fish breeding process, ensuring the stable fry yield of a farm and promoting the healthy development of the fish culture industry; the provided novel fish oxytocic is polypeptide composed of eight amino acids, has no potential safety hazard to fishes and consumers, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of fish oxytocin, and in particular to a novel fish oxytocin and a use method and application thereof. Background Art

[0002] Most bony fish undergo external fertilization, meaning that eggs and sperm combine in water to form fertilized eggs, which then hatch into juveniles. Therefore, ovulation is essential for fish reproduction, and the number of eggs laid is a key indicator of reproductive capacity. The absolute number of eggs a female fish can potentially lay during a breeding season is called "absolute individual fecundity," while the number of eggs a female lays per unit weight or body length during a breeding season is called "relative individual fecundity." These indicators reflect a fish's reproductive capacity and are closely related to population fluctuations and the ability to replenish offspring.

[0003] Different fish species require different external conditions for ovulation, mainly including the following aspects: 1) Water temperature. Fish are very sensitive to water temperature, which directly affects their gonadal development. Water temperatures that are too high or too low can affect their ovulation behavior. The appropriate water temperature range is crucial for fish ovulation. Most fish ovulate most actively at water temperatures between 20-30°C, but the most suitable water temperature is usually between 20-26°C. 2) Light. Light regulates the fish's reproductive cycle by affecting the photoperiod, which in turn affects their ovulation and other reproductive activities. In temperate regions, spring and summer are the main spawning seasons for fish. Extending the photoperiod can promote gonadal development and ovulation. 3) Water quality. The quality of water directly affects the health and reproductive capacity of fish. Clear, unpolluted water is conducive to gonadal development and ovulation. The oxygen content, pH value, water hardness, etc. in the water also need to be within the appropriate range. 4) Nutrition: Adequate nutrition is crucial for fish reproduction. High-quality feed, rich in protein, vitamins, and minerals, can provide fish with the energy and nutrients they need to promote reproductive system development and ovulation. 5) Environment: Providing a safe, stable, and comfortable environment is also crucial for fish reproduction. The aquarium should include ample shelter, plants, and caves to reduce stress and promote reproductive behavior.

[0004] If fish cannot ovulate normally due to unsuitable environmental conditions when their gonads are mature, the eggs will remain in the fish's body. Some will gradually degenerate, while others will pose a variety of health risks. Suffocation and death are among the main hazards of improper egg release. For example, if goldfish cannot release their eggs during spawning, they will suffocate, with a probability of over 70%. They may also die from exhaustion. Furthermore, eggs remaining in the fish's body can cause infection and poisoning, leading to illness or even death. This can also cause economic losses for fish farmers. Therefore, artificial induction of ovulation is an important means to prevent poor ovulation in fish and ensure a stable supply of fish seedlings. Oxytocics are key to successful artificial induction of ovulation. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel fish oxytocin and its use method and application, so as to reduce the adverse effects of the external environment on fish spawning, lower the conditions required during fish reproduction, ensure the stable production of fry in the farm, and provide a novel fish oxytocin without safety hazards.

[0006] To achieve the above-mentioned object, the present invention provides a novel fish oxytocin, which contains a polypeptide as a main component or active ingredient, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0007] Preferably, the amino acid sequence of the polypeptide is shown as SEQ ID NO.2.

[0008] Preferably, the amino acid sequence of the polypeptide has a homology of not less than 85% with the sequence shown in SEQ ID NO. 1.

[0009] A method for using the novel fish oxytocin as described above is as follows: the novel fish oxytocin is dissolved in dimethyl sulfoxide to prepare a mother solution, which is then diluted with fish physiological saline to form a working solution, and the working solution is injected into the body of a fish with mature ovaries.

[0010] Preferably, the concentration of the mother solution is 2 mg / mL and the injection dose is 200 ng oxytocin / g fish.

[0011] Preferably, the injection site is from the base of the fish's pectoral fin into the fish's abdominal cavity.

[0012] Preferably, the injection time is a period of time after the ovarian maturity is observed, which is the time from ovarian maturity to egg maturity as recorded in the prior art. Whether to use daytime injection or nighttime injection depends on the ovulation characteristics of the test fish.

[0013] The invention relates to an application of the novel fish oxytocin as described above in fish farming and in artificially inducing fish ovulation.

[0014] Therefore, the present invention provides a novel fish oxytocin and its use method and application, and its specific technical effects are as follows:

[0015] (1) After intraperitoneal injection of the novel fish oxytocin provided by the present invention, it has a significant induced ovulation effect on zebrafish and can be used to promote ovulation in artificially cultured fish. It is of great significance to reduce the adverse effects of the external adverse environment on fish ovulation, reduce the conditions required for fish reproduction, ensure the stable production of fry in the farm, and promote the healthy development of the fish farming industry;

[0016] (2) The novel fish oxytocin provided by the present invention is a polypeptide composed of 8 amino acids, which poses no safety risks to fish and consumers and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 The zebrafish before injection of oxytocin in Example 2 of the present invention;

[0019] Figure 2 This is a photo of the ovulation pressing operation in Example 2 of the present invention;

[0020] Figure 3 This is a photo of the zebrafish after ovulation in Example 2 of the present invention;

[0021] Figure 4 This is a photo of a zebrafish egg in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0023] In order to make the purpose, technical solutions and advantages of the present application clearer, more thorough and more complete, the technical solutions of the present invention are clearly and completely described below through the accompanying drawings and Examples. The following detailed description is an explanation of the embodiments and is intended to provide further details of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the application belongs.

[0024] The instruments, equipment, and reagents used in the examples were commercially available. Wild-type zebrafish were purchased from the Institute of Hydrobiology, Chinese Academy of Sciences. All experimental procedures were performed in accordance with the Animal Use Regulations of Huazhong Agricultural University (Ethical Approval No. HBAC20091138; Date: November 15, 2009).

[0025] Example 1

[0026] Preparation of a novel fish oxytocin, the specific steps are as follows:

[0027] S11. Send the polypeptide sequence information to the company to synthesize the amino acid sequence as shown in SEQ ID NO.1.

[0028] SEQ ID NO.1:Asp-Tyr-Leu-Gly-Trp-Met-Asp-Phe

[0029] S12. Centrifuge the obtained polypeptide at 10,000 rpm for 10 min, add dimethyl sulfoxide (DMSO) according to the attached instructions to prepare a mother solution with a concentration of 2 mg / mL, and dilute it with fish saline to a working solution with a concentration of 4 μg / mL.

[0030] Example 2

[0031] The effect of the novel fish oxytocin prepared in Example 1 on fish induced fertilization was investigated. Zebrafish were used as experimental materials. Zebrafish are currently the most widely used fish biological model in the world. Zebrafish begin to develop after in vitro fertilization. Under artificial breeding conditions, zebrafish will not ovulate without external stimulation, which makes it easier to count the number of ovulations and avoid errors caused by egg confusion. The specific steps are as follows:

[0032] S21. Existing technology shows that zebrafish become sexually mature 3 months after hatching (zebrafish belly becomes noticeably enlarged). On the second day of the fourth month after hatching (to allow more eggs to mature), 30 zebrafish with mature ovaries were randomly selected (the characteristics of zebrafish with mature ovaries are as follows): Figure 1 The rats were randomly divided into three groups, with 10 rats in each group, two of which were experimental groups and one was the control group.

[0033] S22. Experimental group: 5 μL of the novel fish oxytocin working solution prepared in Example 1 was injected into the abdominal cavity of zebrafish from the base of the pectoral fin the previous night using a 50 μL microsyringe. Control group: an equal amount of physiological saline was injected using the same method.

[0034] After the injection, the zebrafish were placed in a purchased zebrafish recirculating culture system for continued cultivation. The system water temperature was maintained at 28±2°C, pH was 7.5±0.3, and the photoperiod was 14 hours of light (8:00-22:00) and 10 hours of darkness (22:00-8:00).

[0035] S23. After culturing for 12 hours, the next morning, the zebrafish in the experimental and control groups were placed on clean towels, and the zebrafish were ovulated by manually pressing the abdomen. The photos of the zebrafish undergoing ovulation are shown in Figure 2. Figure 2 As shown in the picture of zebrafish after pressing ovulation Figure 3 As shown in the photo, a zebrafish egg row is Figure 4 The number of zebrafish eggs laid in the experimental and control groups was counted manually.

[0036] The statistical results are shown in Table 1.

[0037] Table 1 Statistical results of ovulation volume

[0038]

[0039] As can be seen from Table 1, 10 zebrafish in the experimental group spawned, with an spawning rate of 100%. In contrast, none of the zebrafish in the control group ovulated, indicating that the novel fish oxytocin prepared in Example 1 is effective in promoting zebrafish ovulation.

[0040] Example 3

[0041] A novel fish oxytocin agent was prepared, and the specific steps were exactly the same as those in Example 1, except that the amino acid sequence of the polypeptide was as shown in SEQ ID NO.2.

[0042] SEQ ID NO.2:Asp-Tyr-Val-Gly-Trp-Met-Asp-Phe

[0043] Example 4

[0044] The induced spawning effect of the novel fish oxytocin prepared in Example 3 on zebrafish was investigated, and the specific steps were as follows:

[0045] S41. On the second day of the fourth month after hatching (to allow more eggs to mature), 20 zebrafish with mature ovaries were randomly selected and divided into two groups, with 10 zebrafish in each group: one group was the experimental group and the other group was the control group.

[0046] S42. Experimental group: 5 μL of the novel fish oxytocin working solution prepared in Example 1 was injected into the abdominal cavity of zebrafish from the base of the pectoral fin the previous night using a 50 μL microsyringe. Control group: an equal amount of physiological saline was injected using the same method.

[0047] After the injection, the zebrafish were placed in a purchased zebrafish recirculating culture system for continued cultivation. The system water temperature was maintained at 28±2°C, pH was 7.5±0.3, and the photoperiod was 14 hours of light (8:00-22:00) and 10 hours of darkness (22:00-8:00).

[0048] S43. After culturing for 12 hours, the next morning, the zebrafish in the experimental and control groups were placed on clean towels, and the zebrafish were manually pressed on the abdomen to ovulate. The number of eggs ovulated by the zebrafish in the experimental and control groups was counted manually.

[0049] The statistical results are shown in Table 2.

[0050] Table 2 Statistical results of ovulation volume

[0051]

[0052]

[0053] As can be seen from Table 2, 8 zebrafish in the experimental group spawned, with an spawning rate of 8 / 10 × 100% = 80%. In contrast, none of the zebrafish in the control group ovulated, indicating that the novel fish oxytocin prepared in Example 3 is effective in promoting zebrafish ovulation.

[0054] Therefore, after intraperitoneal injection of the novel fish oxytocin provided by the present invention, it has a significant induced ovulation effect on zebrafish, can be used to promote ovulation in artificially cultured fish, and is of great significance for reducing the adverse effects of adverse external environments on fish ovulation, lowering the conditions required during fish reproduction, ensuring the stable production of fry in farms, and promoting the healthy development of the fish farming industry. The novel fish oxytocin provided is a polypeptide composed of 8 amino acids, has no safety hazards to fish and consumers, and has broad application prospects.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A novel fish oxytocin, characterized in that: The oxytocic agent has a polypeptide as a main or active ingredient, and the amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A novel fish oxytocin according to claim 1, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.

2.

3. A novel fish oxytocin according to claim 1, characterized in that: The amino acid sequence of the polypeptide has a homology of not less than 85% with the sequence shown in SEQ ID NO.

1.

4. A method for using the novel fish oxytocin according to any one of claims 1 to 3, characterized in that: The method is as follows: the new fish oxytocin is dissolved in dimethyl sulfoxide to prepare a mother solution, which is then diluted with fish physiological saline to form a working solution, and the working solution is injected into the body of a fish with mature ovaries.

5. The method for using the novel fish oxytocin according to claim 4, characterized in that: The concentration of the mother solution was 2 mg / mL, and the injection dose was 200 ng oxytocin / g fish.

6. The method for using the novel fish oxytocin according to claim 4, characterized in that: The injection site is from the base of the fish's pectoral fin into the fish's abdominal cavity.

7. The method for using the novel fish oxytocin according to claim 4, characterized in that: The injection time is a period of time after the ovarian maturity is observed. The period of time is the time from ovarian maturity to egg maturity as recorded in the prior art. Whether to use daytime injection or nighttime injection is determined by the ovulation characteristics of the test fish.

8. Use of the novel fish oxytocin according to any one of claims 1 to 3 in fish farming, characterized in that: Application in artificial induction of fish ovulation.

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