Method for artificially obtaining fertilized eggs of rana nigromaculata

Abstract

The invention relates to the technical field of frog breeding, in particular to a method for artificially obtaining fertilized eggs of Pleurotus nigromaculatus. According to the method, high-protein feed and tocopherol are fed to female breeding frogs and male breeding frogs of the rana nigromaculata, and the female breeding frogs and the male breeding frogs are paired after sexual maturity. Practical breeding and oviposition experiment verification shows that when 0.6-0.8 g of tocopherol is added into each 1g of feed for mixed feeding of the paphiophyllum nigromaculatum in the sexual development stage, the sexual maturity time can be remarkably shortened by 14.3% compared with that of a group without addition, the sexual maturity of the paphiophyllum nigromaculatum is promoted by adding the tocopherol preparation, and the sexual maturity rate of the paphiophyllum nigromaculatum is improved. According to the method, the hormone mixed liquid is injected into the abdominal cavity to induce spawning, the spawning success rate is increased, fertilized eggs are obtained, the method is effective and feasible, and compared with a traditional breeding spawning method, the method has remarkable advantages and can be applied to actual production research.

Description

Technical Field

[0001] This invention relates to the field of frog farming technology, and in particular to a method for artificially obtaining fertilized eggs of the black-spotted lateral fold frog. Background Technology

[0002] The black-spotted lateral frog (Pelophylax nigromaculatus), belonging to the genus Pelophylax in the family Ranidae of the order Anura, is one of the most widely distributed amphibians in my country. It inhabits paddy fields, ponds, lakes, marshes, and mountains below 2200 meters in altitude in plains or hilly areas, possessing abundant germplasm resources. The black-spotted lateral frog is a commonly used research animal in laboratories due to its diploid chromosomes, metamorphosis from aquatic to terrestrial life, and its advantages such as large numbers, ease of capture and breeding, non-toxicity, and low price. It is an excellent animal material among amphibians and has significant value in research on basic traits. At the same time, the black-spotted lateral frog is also an important farmed economic frog species in my country, and its market share in the edible frog market has been continuously expanding and increasing in recent years.

[0003] However, the breeding period of the Black-spotted Frog (Pterygodon nigra) in its natural state is quite long. Currently, in actual aquaculture production, the breeding of the Black-spotted Frog typically involves a life cycle process including hibernation, emergence from hibernation, and egg-laying. From February to May each year, as temperatures rise, a large number of new eggs hatch. After 5 to 6 months of growth, development, and sexual maturity, they enter a 3 to 4 month hibernation period from October to November, only resuming the next breeding cycle in February or March of the following year. Therefore, in its natural state, the Black-spotted Frog's egg-laying cycle is only once a year, and the egg-laying time is relatively concentrated, with only a 3 to 5 month window within a year for obtaining fertilized eggs, which is insufficient to meet the needs of relevant scientific research. Therefore, how to continuously and rapidly obtain multiple batches of fertilized eggs in large quantities to provide excellent experimental materials for subsequent CRISPR / Cas9 gene editing and other related research has become a major problem that urgently needs to be solved in the breeding and propagation of the Black-spotted Frog. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a method for artificially obtaining fertilized eggs of the black-spotted frog, which enables artificial, controllable, and rapid acquisition of fertilized eggs.

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

[0006] This invention provides a method for artificially obtaining fertilized eggs of the black-spotted frog, comprising the following steps:

[0007] 1) Feeding female and male breeding frogs of the Black-spotted Frog with high-protein feed and tocopherols resulted in sexually mature female and male breeding frogs.

[0008] The feeding amount of the high-protein feed is 3-5g per 50g frog per day;

[0009] The feeding dosage of tocopherol is: 0.6-0.8g of tocopherol added per 1g of high-protein feed;

[0010] 2) The sexually mature female and male frogs obtained in step 1) were treated with hormones, including luteinizing hormone-releasing hormone A2 and human chorionic gonadotropin (HCG).

[0011] 3) The female and male frogs treated with hormones were paired to lay eggs, and fertilized eggs of the black-spotted lateral fold frog were obtained.

[0012] Preferably, in step 1), the female and male breeding frogs of the Black-spotted Side-folded Frog are raised in an aquatic ecological tank. The aquatic ecological tank is configured with water and land in an area ratio of 3:7. The bottom of the land is filled with a layer of filter media 2-3 cm thick, and pebbles are laid on top of it. The individual pebbles have a diameter of 0.5-1.0 cm and a cumulative height of 3-5 cm.

[0013] The dimensions of the aquatic ecological tank are: length 0.8m, width 0.6m, height 0.6m, water height 8-12cm, and land height 10-15cm.

[0014] Preferably, the stocking density of female breeding frogs in the aquatic ecological tank is 15-20 per tank, and the stocking density of male breeding frogs is 20-25 per tank.

[0015] Preferably, the ambient temperature in the aquatic-terrestrial ecological tank is controlled at 24–26°C.

[0016] Preferably, pairings are performed at a ratio of 1:1 between sexually mature female and male breeding frogs in each spawning tank.

[0017] Preferably, the spawning tank has the following specifications: 0.6m*0.4m*0.4m. The tank is filled with clear aerated water to a depth of 5-10cm. The water is free of impurities and turbidity, and the water temperature is 22-24℃. The top of the tank is sealed with a transparent mesh to ensure that the paired adult frogs cannot jump out. At the same time, the situation inside the tank can be clearly observed. A monitoring camera is installed above the spawning tank to remotely monitor the mating and spawning in real time.

[0018] Preferably, the temperature of the paired water is controlled at 22-24°C.

[0019] Preferably, in step 2), the hormone is treated in the form of a hormone solution, with each 10 ml of hormone solution containing 0.2 mg of luteinizing hormone-releasing hormone A2 and 0.2 mg of human chorionic gonadotropin (HCG); 200 μl of hormone solution is used for every 50 g of breeding frogs.

[0020] The beneficial effects of this invention are:

[0021] Actual breeding and egg-laying experiments have verified that feeding black-spotted lateral fold frogs with feed containing 0.6–0.8 g of tocopherol per 1 g of feed significantly shortens the sexual maturity time by 14.3% compared to the group without tocopherol. Promoting sexual maturity through the addition of tocopherol, followed by induced spawning via intraperitoneal injection of a hormone mixture, improves the egg-laying success rate and the acquisition of fertilized eggs. This method is effective and feasible, showing significant advantages over traditional breeding and egg-laying methods, and can be applied to practical production research. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0023] Figure 1 These are sexually mature Black-spotted Frogs; the one on the left is a female, and the one on the right is a male.

[0024] Figure 2 An oviposition device for the black-spotted lateral fold frog;

[0025] Figure 3 The effect of adding tocopherol to feed on the sexual maturity time of the black-spotted lateral fold frog;

[0026] Figure 4 The effect of A2-HCG mixed hormone injection on oviposition success rate. Detailed Implementation

[0027] This invention provides a method for artificially obtaining fertilized eggs of the black-spotted frog, comprising the following steps:

[0028] 1) Feeding female and male breeding frogs of the Black-spotted Frog with high-protein feed and tocopherols resulted in sexually mature female and male breeding frogs.

[0029] The feeding amount of the high-protein feed is 3-5g per 50g frog per day;

[0030] The feeding dosage of tocopherol is: 0.6-0.8g of tocopherol added per 1g of high-protein feed;

[0031] 2) The sexually mature female and male frogs obtained in step 1) are treated with hormones, including luteinizing hormone-releasing hormone A2 and human chorionic gonadotropin (HCG).

[0032] 3) The female and male frogs treated with hormones were paired to lay eggs, and fertilized eggs of the black-spotted lateral fold frog were obtained.

[0033] This invention involves feeding female and male *Rana maculatus* (black-spotted lateral fold frog) with a high-protein diet and tocopherol to obtain sexually mature female and male frogs. The feeding amount of the high-protein diet is 3-5g per 50g of frog per day. The feeding amount of tocopherol is 0.6-0.8g of tocopherol added to every 1g of high-protein diet.

[0034] In this invention, the selection criteria for female and male breeding frogs of the Black-spotted Side-folded Frog are: healthy, disease-free (no signs of intoxication, head tilting, or limb weakness), with suitable body shape and a full abdomen. The criteria for sexually mature female breeding frogs are: pressing the abdomen and sides of the female frog with fingers reveals a distinct, hard, and firm lump, indicating it is filled with mature egg cells; the criteria for sexually mature male breeding frogs are: gently pinching the sides of the male frog's abdomen with two fingers reveals bulging external vocal sacs and a loud call. The high-protein feed used in this invention is commercially available from Jiangmen Xinjian Feed Co., Ltd., produced in Jiangmen City, Guangdong Province, with product model Xinjian Peptide (3#A146603). This is a functional compound feed for frogs, composed of high-quality imported fishmeal, refined deep-sea fish oil, soybean meal, peanut meal, wheat flour, soybean lecithin oil, immunomodulators, traditional Chinese medicine extracts, calcium dihydrogen phosphate, multiple vitamins, complex calcium substances, antioxidants, and antifungal agents. It contains 40.0% crude protein, 2.2% lysine, 4.0% crude fat, 5.0% crude fiber, and 15.0% crude ash.

[0035] In this invention, both female and male breeding frogs of the Black-spotted Side-folded Frog are raised in an aquatic ecological tank. The preferred specifications of the aquatic ecological tank are: a water-to-land area ratio of 3:7; a 2-3 cm thick layer of filter media at the bottom of the land, topped with pebbles (each pebble 0.5-1.0 cm in diameter, accumulating to a height of 3-5 cm); and dimensions of the aquatic ecological tank of 0.8 m length, 0.6 m width, and 0.6 m height, with a water depth of 8-12 cm and a land depth of 10-15 cm. Smooth, edgeless stones should be selected to prevent adult frogs from bumping into and damaging their body surface tissues. Preferably, a feeding dish containing tocopherol and high-protein feed is placed on the land; a water pump area is located at the angle between the land and the tank wall, drawing water through a conduit to the water for internal circulation, achieving filtration and purification. In this invention, the preferred stocking density of female breeding frogs in the aquatic ecological tank is 15-20 frogs / tank, and the preferred stocking density of male breeding frogs is 20-25 frogs / tank. In this invention, the ambient temperature in the aquatic ecological tank is preferably controlled at 24-26°C.

[0036] This invention involves treating sexually mature female and male breeding frogs with hormones, including luteinizing hormone-releasing hormone A2 and human chorionic gonadotropin (HCG). Preferably, the hormones are administered as a solution containing 0.2 mg of luteinizing hormone-releasing hormone A2 and 0.2 mg of HCG per 10 ml of physiological saline; 200 μl of the hormone solution is preferably used per 50 g of breeding frogs. The hormone solution is preferably injected into both female and male breeding frogs via abdominal injection.

[0037] This invention involves pairing female and male frogs treated with hormones to produce fertilized eggs of the black-spotted lateral fold frog.

[0038] In this invention, mating is preferably carried out at a ratio of 1:1 between sexually mature female and male breeding frogs in each spawning tank. The spawning tank has dimensions of 0.6m x 0.4m x 0.4m, filled with clear, aerated water to a depth of 5-10cm. The water is free of impurities and turbidity, and the water temperature is 22-24℃. The top of the tank is sealed with a transparent mesh to ensure that the mated frogs cannot jump out, while allowing clear observation of the tank's interior. A monitoring camera is mounted above the spawning tank for remote, real-time monitoring of mating and egg-laying. In this invention, the water temperature for mating is preferably controlled at 22-24℃.

[0039] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] A method for artificially obtaining fertilized eggs of the black-spotted lateral fold frog, comprising the following steps:

[0042] (1) Indoor breeding and maturation methods for adult frogs: mainly for female and male breeding frogs, healthy and disease-free (no rotten mouth, wry head, no weak limbs, etc.), with suitable body size and full abdomen should be selected to facilitate sexual maturity.

[0043] Adult frogs are raised in a hydroponic terrarium. The hydroponic terrarium is set up as follows: the water and land areas are configured in a 3:7 ratio. The bottom of the land area is filled with a 2.0-3.0cm thick layer of filter media, on which fine pebbles are laid flat. The individual pebbles are about 0.5-1.0cm in diameter, and the accumulated height is 3.0-5.0cm. Smooth, edgeless stones should be selected to avoid damaging the frogs' body surface tissues. A feeding dish is placed on the land area to hold spawning stimulants and feed. A water pump area is set up at the angle between the land area and the tank wall, and a water pump is connected to the water area through a pipe to circulate the water in the tank, achieving the functions of filtration and purification.

[0044] The optimal room temperature for inducing maturation in adult frogs is 24–26℃. A mixture of high-protein feed and tocopherol preparation is used for this purpose. The specific addition method is as follows: Based on the number of frogs in the tank (stocking density of female breeding frogs is 15.0–20.0 individuals / tank, and the stocking density of male breeding frogs is 20.0–25.0 individuals / tank) and the weight of each frog, feed high-protein feed at an average rate of 3.0–5.0g / day per 50.0g of frog. Simultaneously, add 0.6g of tocopherol per 1.0g of high-protein feed. Ten groups (ten replicates) were selected.

[0045] (2) Selection method for sexually mature frogs: Adult frogs in breeding terrariums can reach sexual maturity significantly faster after being treated with tocopherol. Specific criteria for selecting sexually mature frogs are as follows ( Figure 1 Specifically: Male frog: Gently pinch the sides of the male frog's abdomen with two fingers, and you can see the external vocal sacs bulge and the call is loud; Female frog: Press the abdomen and sides of the female frog with your fingers, and you can feel obvious hard and solid lumps, which are filled with mature egg cells.

[0046] (3) Pairing and egg laying of adult frogs: Select male and female adult frogs with good body condition and sexual maturity to pair up. Inject the sexually mature female and male frogs with mixed hormones respectively. Hormone preparation: Use 15ml enzyme-free centrifuge tubes to prepare the injection mixture by adding 0.2mg of A2 and HCG hormones to every 10ml of physiological saline, and store it in a refrigerator at 4℃ for later use.

[0047] The injection method was as follows: adult frogs were induced to mate and lay eggs by injecting a mixture of luteinizing hormone-releasing hormone A2 and human chorionic gonadotropin (HCG). Male and female adult frogs in good condition and sexually mature were selected and pretreated at 9-10 PM, with the hormone mixture injected using a sterile syringe to induce spawning. The injection mixture, stored at 4℃, was then administered intraperitoneally to both male and female frogs at a dosage of 200 μL per 50g of adult frog. After injection, the male and female frogs were placed separately.

[0048] The following morning, between 9 and 10 a.m., the induced adult frogs were placed into the spawning tanks for mating, with a female-to-male ratio of 1:1 per tank. The ideal water temperature for mating was 22-24°C. During mating, environmental factors should be avoided, and human disturbance to the room should be minimized. Spawning usually occurs within 1-2 hours after successful mating. A total of 5 pairs (5 parallel pairs) were selected.

[0049] The breeding and spawning tank for male and female frogs measures 0.6m x 0.4m x 0.4m. It contains 5-10cm of clear, aerated water, free of impurities and turbidity, at a suitable temperature of 22-24℃. The top of the tank is sealed with a transparent mesh to prevent paired adult frogs from jumping out while allowing clear observation of the tank's interior. A monitoring camera is mounted above the spawning tank for remote, real-time monitoring of mating and egg-laying.

[0050] Comparative Example 1

[0051] The difference from Example 1 is that no tocopherol was added during feeding, and the time to reach sexual maturity was compared.

[0052] Comparative Example 2

[0053] The difference from Example 1 is that no mixed hormone was injected into the abdomen to compare the success rate of egg spawning.

[0054] from Figure 3 The results show that, compared with the control group that was not fed with tocopherol, feeding with tocopherol significantly shortened the sexual maturity time of the black-spotted frog, indicating that adding tocopherol helps promote the sexual maturity of adult frogs.

[0055] from Figure 4 As can be seen, compared with the control group that did not receive intraperitoneal injection, the mixed injection of A2 and HCG hormones in the egg-laying experiment can greatly increase the egg-laying success rate by more than 50%, effectively promoting the egg-laying of the black-spotted frog.

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for artificially obtaining fertilized eggs of the black-spotted lateral-fold frog, characterized in that, Includes the following steps: 1) Feeding female and male breeding frogs of the Black-spotted Frog with high-protein feed and tocopherols resulted in sexually mature female and male breeding frogs. The feeding amount of the high-protein feed is 3-5g per 50g frog per day; The feeding dosage of tocopherol is: 0.6-0.8g of tocopherol added per 1g of high-protein feed; 2) The sexually mature female and male frogs obtained in step 1) were treated with hormones, including luteinizing hormone-releasing hormone A2 and human chorionic gonadotropin (HCG). 3) The female and male frogs treated with hormones were paired to lay eggs, and fertilized eggs of the black-spotted lateral fold frog were obtained.

2. The method according to claim 1, characterized in that, In step 1), the female and male breeding frogs of the Black-spotted Side Fold Frog are raised in an aquatic ecological tank. The specifications of the aquatic ecological tank are as follows: the area ratio of water to land is 3:

7. The bottom of the land is filled with a layer of filter media 2-3cm thick, and pebbles are laid flat on top of it. The individual diameter of the pebbles is 0.5-1.0cm, and the cumulative height is 3-5cm. The dimensions of the aquatic ecological tank are: length 0.8m, width 0.6m, height 0.6m, water height 8-12cm, and land height 10-15cm.

3. The method according to claim 2, characterized in that, The stocking density of female breeding frogs in the aquatic ecological tank is 15-20 per tank, and the stocking density of male breeding frogs is 20-25 per tank.

4. The method according to claim 2, characterized in that, The ambient temperature in the aquatic-terrestrial ecological tank is controlled at 24–26°C.

5. The method according to claim 1, characterized in that, Pairing is performed according to a 1:1 ratio of sexually mature female to male breeding frogs in each spawning tank.

6. The method according to claim 5, characterized in that, The spawning tank has the following dimensions: 0.6m*0.4m*0.4m. It is filled with clear, aerated water to a depth of 5-10cm. The water is free of impurities and turbidity, and the water temperature is 22-24℃. The top of the tank is sealed with a transparent mesh to ensure that the paired adult frogs cannot jump out, while allowing clear observation of the situation inside the tank. A monitoring camera is installed above the spawning tank to remotely monitor the mating and spawning in real time.

7. The method according to claim 1 or 5, characterized in that, The temperature of the paired water is controlled at 22-24℃.

8. The method according to claim 1, characterized in that, In step 2), the hormones are treated in the form of a hormone solution, with each 10 ml of hormone solution containing 0.2 mg of luteinizing hormone-releasing hormone A2 and 0.2 mg of human chorionic gonadotropin (HCG); 200 μl of hormone solution is used for every 50 g of breeding frogs.

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