A method for the artificial collection and preservation of mature pomfret eggs

Abstract

This invention relates to the field of aquatic animal farming, and particularly to a method for the artificial collection and preservation of mature pomfret eggs. This method allows for the acquisition of a large number of mature pomfret eggs under fully artificial conditions and their preservation in vitro for a relatively long period, providing support for research on pomfret fertilization biology, fully artificial reproduction technology, and embryonic development.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal farming, and in particular to a method for the artificial collection and preservation of mature pomfret eggs. Background Technology

[0002] Golden pomfret is one of the major species in tropical marine aquaculture in my country. Currently, it faces serious bottlenecks restricting its seed industry development, including low artificial breeding efficiency and a lack of superior new varieties. Artificial spawning induction in golden pomfret is inefficient, and fully artificial breeding technology has not yet been fully mastered. Currently, the main method involves indiscriminately injecting oxytocin into the parent stock followed by natural mating and fertilization. Because the maturity of the eggs cannot be determined, the spawning induction efficiency is only about 10%, and the fertilization rate is less than 50%. To increase spawning, multiple spawning induction methods are often used on the parent stock, which not only causes significant damage to the breeding parents but also doubles the breeding costs due to the large-scale use of oxytocin. Furthermore, the inability to obtain artificially fertilized eggs hinders a series of scientific research efforts on golden pomfret reproductive biology and embryonic development biology, severely limiting the development of the golden pomfret industry.

[0003] Currently, there is no mature technology for collecting and preserving eggs in golden pomfret. Although egg preservation research has been conducted on many fish species, decades of research have resulted in a series of mature artificial maturation and spawning techniques for major freshwater fish. However, research on artificial breeding technology for golden pomfret is still in its early stages, with a significant lack of research on methods and techniques for sex determination and gonadal maturity detection. Therefore, to further promote the development of the golden pomfret industry, it is urgent to establish an artificially controllable breeding technology, and the artificial collection, preservation, and application of mature eggs are the foundation of fully artificial breeding technology. Summary of the Invention

[0004] In view of this, the present invention provides a method for the artificial collection and preservation of mature pomfret eggs. This method addresses the problem of outdated artificial breeding technology for pomfret by detecting egg maturity and then artificially collecting, preserving, and using the eggs, thereby greatly improving the efficiency of pomfret breeding and scientific research.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for obtaining mature eggs from golden pomfret, comprising the following steps:

[0007] Step (1): Determine the maturity of the gonads of the golden pomfret parent fish. If the gonads are mature, artificial spawning will be induced.

[0008] Step (2): Obtain the eggs of the golden pomfret parent fish and determine the maturity of the eggs. If the eggs are fully mature, artificial egg collection will be carried out to obtain mature golden pomfret eggs.

[0009] In some specific embodiments of the present invention, the method step (2) above further includes a step of preserving the mature pomfret eggs, wherein the preservation conditions include:

[0010] The temperature refers to the temperature of the water in which the parent fish are located;

[0011] Maintain ventilation to preserve oxygen levels.

[0012] In some specific embodiments of the present invention, the preservation conditions described in the above method further include increasing air humidity.

[0013] In some specific embodiments of the present invention, the criteria for judging the gonadal development maturity of the golden pomfret parent fish in the above method step (1) include: negative pressure suction of the eggs of the golden pomfret parent fish; if eggs flow out, it indicates that the gonadal development is mature.

[0014] In some specific embodiments of the present invention, the artificial induced labor described above includes injecting fish chorionic gonadotropin and fish luteinizing hormone-releasing hormone.

[0015] In some specific embodiments of the present invention, the injection described above is performed in two sessions, with each session 24 hours apart.

[0016] In some specific embodiments of the present invention, the injection dose of the first injection in the above method is 300 U / kg of fish human chorionic gonadotropin and 2 μg / kg of fish luteinizing hormone-releasing hormone.

[0017] The second injection dose was 200 U / kg of fish chorionic gonadotropin and 2 μg / kg of fish luteinizing hormone-releasing hormone.

[0018] In some specific embodiments of the present invention, the criteria for determining the maturity of the egg in the above method include: if the egg is separated and transparent, it indicates that the egg is fully mature.

[0019] In some specific embodiments of the present invention, step (2) of the above method further includes massaging the abdomen of the golden pomfret parent fish before artificial spawning.

[0020] In some specific embodiments of the present invention, the above method step (2) involves artificially producing eggs by squeezing.

[0021] The squeezing includes: clamping the two sides of the abdomen of the golden pomfret parent fish and squeezing from the front to the vicinity of the cloaca, while simultaneously clamping the two sides of the abdomen from behind the cloaca and squeezing forward to allow the eggs to be discharged from the cloaca.

[0022] In some specific embodiments of the present invention, the above method step (2) artificial oviposition also includes the step of inserting an oviposition tube into an oviposition tube to assist oviposition.

[0023] The method of the present invention has the following effects:

[0024] 1. Method for collecting immature eggs: This invention uses a negative pressure method to collect immature eggs from golden pomfret before and after spawning to determine the maturity of the eggs. This method has not been applied to other fish species and is the first of its kind in this invention.

[0025] 2. Artificial Collection of Mature Golden Pomfret Eggs: After reaching sexual maturity, only a very small number of female golden pomfret exhibit abdominal swelling; most show only slight distension. Therefore, it is difficult to collect mature eggs using conventional squeezing methods. This invention assesses egg maturity beforehand, selects mature parent fish, massages their abdomens until softened, and then applies bidirectional squeezing. An egg collection tube is used to assist in egg collection, successfully obtaining a large number of mature golden pomfret eggs. This is a pioneering achievement both domestically and internationally.

[0026] 3. Preservation of Mature Pomfret Eggs: Currently, most fish egg preservation methods are conventional, involving placing the eggs in a container and storing them in a refrigerator or insulated box at around 4°C. However, pomfret are warm-water fish, and their mature eggs cannot withstand low temperatures, and they are extremely intolerant of low-oxygen environments. Therefore, this invention uses the same water temperature as the pomfret spawning pond and employs a humidified gas aeration method for preservation, greatly improving the preservation time and fertilization rate of pomfret eggs. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0028] Figure 1 The maturation of pomfret oocytes is shown in the diagram, where A represents immature oocytes and B represents mature oocytes.

[0029] Figure 2 This shows the artificial collection of mature eggs from golden pomfret. Detailed Implementation

[0030] This invention discloses a method for the artificial collection and preservation of mature pomfret eggs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] To address the problems described in the background art, this invention provides a method for the artificial collection, preservation, and application of golden pomfret eggs. This method can obtain a large number of mature golden pomfret eggs under fully artificial conditions and preserve them in vitro for a long time, providing support for research on golden pomfret fertilization biology, fully artificial reproduction technology, and embryo development.

[0032] The method for artificial collection, preservation and application of mature pomfret eggs provided by this invention will provide technical support for the establishment of fully artificial breeding technology for pomfret.

[0033] A method for the artificial collection, preservation, and application of mature pomfret eggs, including:

[0034] (1) Under artificial breeding conditions, select sexually mature female parent fish that are 5 years old or older and weigh more than 8 kg;

[0035] (2) During the breeding season, the sexually mature female parent fish are fortified with nutrition. One month before breeding, frozen squid and fresh fish are fed to promote ovarian development.

[0036] (3) Before artificially injecting oxytocin, check the maturity of the broodstock's gonads. The specific method is to use an egg collection device, lubricate the egg collection tube with petroleum jelly, and insert it into the ovary through the pomfret's spawning pore, stopping when it is inserted about 5-10 cm;

[0037] (4) Pull the oocyte collection device to create negative pressure and observe whether any eggs flow out. If any eggs flow out, it indicates that the gonads are mature and artificial labor can be induced.

[0038] (5) If no eggs are released, slowly move the egg collection tube and observe whether any eggs are released. If eggs are released, it indicates that the gonads are mature. If no eggs are released, it indicates that the gonads are not mature enough and artificial induction of labor is not possible. Further cultivation is still needed.

[0039] (6) Selected mature female golden pomfret parents were artificially induced to spawn. The spawning hormones were fish human chorionic gonadotropin (HCG, Ningbo No. 2 Hormone Factory) and fish luteinizing hormone-releasing hormone (LRH-A2, Ningbo No. 2 Hormone Factory). The mixture was injected twice. The first injection dose was 300 U / kg HCG and 2 μg / kg LRH-A2. The second injection was given 24 hours later, with a dose of 200 U / kg HCG and 2 μg / kg LRH-A2.

[0040] (7) Twelve hours after the second injection, the development of the female fish's gonads was checked. At this time, the abdomen began to swell;

[0041] (8) Select parent fish with obvious abdominal swelling, collect eggs using an egg collector and observe. When all eggs are observed to be separated and transparent, it indicates that the eggs are fully mature and can be collected artificially.

[0042] (9) Unlike other fish, the golden pomfret has a relatively hard abdomen even during the breeding season. Therefore, it is necessary to massage and rub the abdomen before artificial egg collection to soften it and make it easier to squeeze out the eggs.

[0043] (10) When collecting eggs, place the parent plant flat on the egg collection platform, hold the abdomen on both sides with your right hand, and squeeze from the front to the back. When you squeeze to the vicinity of the cloaca, hold the abdomen on both sides from the back of the cloaca and squeeze forward. You can see the eggs being discharged from the cloaca. Collect the eggs with the prepared egg collector.

[0044] (11) If no eggs are produced after squeezing, an egg collection tube can be inserted into the oviposition tube to assist in ovulation;

[0045] (12) Measure the water temperature of the parent fish pond, adjust the egg preservation box to the same temperature as the water temperature, spread the squeezed eggs flat in the egg preservation box, with each layer not exceeding 2cm in thickness, and oxygenate the egg preservation box through an external air pump.

[0046] (13) To prevent the upper layer of eggs from drying out, the gas from the external air pump will be moistened with sterile water before entering the egg storage box to ensure a moist environment in the box.

[0047] (14) Under these conditions, the fertilization rate can reach more than 80% after the eggs are preserved for 3 hours.

[0048] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0049] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0050] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0051] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0052] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

[0053] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0054] The present invention will be further illustrated below with reference to the embodiments:

[0055] Example 1: Maturity check of golden pomfret eggs and artificial egg collection

[0056] 1. Inducing spawning in parent fish

[0057] Female golden pomfret parent fish aged 5 years or older and weighing over 8 kg were selected for nutritional fortification. Before artificially injecting oxytocin, the maturity of the parent fish's gonads was checked. If the gonads were mature, they could be placed in the spawning pond for artificial spawning induction. Twelve hours after the second spawning induction, the gonad development of the female fish was checked again.

[0058] 2. Determining the maturity of golden pomfret eggs

[0059] Twelve hours after induced labor, oocytes are retrieved using an oocyte retrieval device, and their maturity is assessed. When all oocytes are observed to be separated and translucent, they indicate full maturity and are ready for artificial oocyte retrieval. Immature oocytes, such as… Figure 1 As shown in A, a mature egg cell is like... Figure 1 As shown in B in the diagram.

[0060] 3. Artificial collection of golden pomfret eggs

[0061] When collecting eggs, place the parent plant flat on the egg collection table, hold the abdomen on both sides with your right hand, and squeeze from the front to the back. When you reach the vicinity of the cloaca, use your left hand to hold the abdomen on both sides from behind the cloaca and squeeze forward. You can see the eggs being released from the cloaca. Collect the eggs with the prepared egg collector. Figure 2These are artificially collected mature golden pomfret eggs. The success rate and quantity of eggs obtained using different extraction methods are shown in Table 1. The criterion for judging the success rate of egg extraction is: being able to extract more than 50 mL of eggs is considered successful.

[0062] Table 1: Success rate and number of eggs obtained by different squeezing methods

[0063] Group Number of eggs retrieved (mL) Success rate (%) biaxial extrusion 300 100 Squeeze from head to tail 25 30 Squeeze from tail to head 0 0

[0064] Example 2: Preservation of Golden Pomfret Eggs

[0065] 1. Preservation of golden pomfret eggs in a preservation box

[0066] Set the egg preservation chamber to the same temperature as the water. Spread the extruded eggs evenly in the chamber, with each layer not exceeding 2 cm in thickness. Aerate the egg preservation chamber using an external air pump. To prevent the upper layer of eggs from drying out, humidify the air in the external air pump with sterile water before it enters the chamber, ensuring an air humidity of no less than 95% and maintaining a humid environment within the chamber.

[0067] 2. Artificial insemination of golden pomfret eggs

[0068] Artificial insemination was performed on eggs preserved in a golden pomfret oocyte preservation incubator. Insemination was performed after 1, 2, 3, and 4 hours of preservation. The fertilization rate was 84.14% after 1 hour, 86.48% after 2 hours, 82.23% after 3 hours, and 75.98% after 4 hours. The fertilization rate remained above 80% even after 3 hours of preservation. A control group preserved under low-temperature, normoxic conditions showed a significantly lower fertilization rate. Specific data are shown in Table 2.

[0069] Table 2: Fertilization rate of golden pomfret eggs after different storage times

[0070]

[0071] Example 3: The effect of oocyte storage temperature on fertilization rate

[0072] Oocytes were collected according to Example 1, and the experimental group was preserved according to the method described in Example 2. Oocytes in the experimental group were collected and preserved at 25°C, while those in the control group were preserved at 15°C and 30°C, respectively. Artificial insemination was performed after 1, 2, 3, and 4 hours of preservation. The fertilization rate was 84.14% after 1 hour, 86.48% after 2 hours, 82.23% after 3 hours, and 75.98% after 4 hours. The fertilization rate remained above 80% even after 3 hours of preservation. The control group, preserved under low-temperature and normoxic conditions, showed a significantly lower fertilization rate after preservation compared to the experimental group. Specific data are shown in Table 3.

[0073] Table 3: Fertilization rate of golden pomfret eggs after different storage times

[0074]

[0075] Example 4: Effects of oocyte storage temperature and water temperature difference on fertilization rate

[0076] Oocytes were collected according to Example 1. The experimental groups were preserved using the method described in Example 2. The control group had oocytes collected and preserved at 24°C. Experimental group 1 was preserved at 28°C, 4°C higher than the control group; experimental group 2 at 32°C, 8°C higher than the control group; experimental group 3 at 20°C, 4°C lower than the control group; and experimental group 4 at 16°C, 8°C lower than the control group. Artificial insemination was performed after 1, 2, 3, and 4 hours of preservation. The results showed that water temperatures higher or lower than the preservation temperature affected the fertilization rate; therefore, oocytes should be preserved at the same temperature as the water. Specific data are shown in Table 4.

[0077] Table 4: Fertilization rate of golden pomfret eggs after different storage times

[0078]

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for obtaining mature eggs from golden pomfret, characterized in that, Includes the following steps: Step (1): Determine the maturity of the gonads of the golden pomfret parent fish. If the gonads are mature, artificial spawning will be induced. Step (2): Obtain the eggs of the golden pomfret parent fish and determine the maturity of the eggs. If the eggs are fully mature, massage the abdomen of the golden pomfret parent fish and collect the eggs artificially by squeezing to obtain mature golden pomfret eggs. Step (3): Preserve the mature pomfret eggs, wherein the preservation conditions include: Measure the water temperature of the parent fish pond, adjust the egg preservation box to the same temperature as the water, and use an external air pump to aerate the eggs. Before the air from the external air pump enters the egg preservation box, it is humidified with sterile water, and the air humidity is not less than 95%. The criteria for judging the gonadal maturity of the golden pomfret parent fish include: negative pressure suction of the eggs of the golden pomfret parent fish; if eggs flow out, it indicates that the gonads are mature; the criteria for judging the maturity of the eggs include: if the eggs are separated and transparent, it indicates that the eggs are fully mature. The squeezing includes: clamping the two sides of the abdomen of the golden pomfret parent fish and squeezing from the front to the vicinity of the cloaca, while simultaneously clamping the two sides of the abdomen from behind the cloaca and squeezing forward to allow the eggs to be discharged from the cloaca.

2. The method as described in claim 1, characterized in that, The artificial induced labor involves injecting fish-specific human chorionic gonadotropin and fish-specific luteinizing hormone-releasing hormone.

3. The method as described in claim 2, characterized in that, The injection was administered in two doses, with a 24-hour interval between each dose. The first injection dose was 300 U / kg of fish human chorionic gonadotropin and 2 μg / kg of fish luteinizing hormone-releasing hormone. The second injection dose was 200 U / kg of fish chorionic gonadotropin and 2 μg / kg of fish luteinizing hormone-releasing hormone.

4. The method according to any one of claims 1 to 3, characterized in that, Step (2) Artificial egg retrieval also includes the step of inserting an egg retrieval tube into the ovulation tube to assist ovulation.

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