Artificial propagation technique of cultured forest frog

With the continuous expansion of forest frog breeding scale, more and more forest frog seedlings are needed, which can not be met by transplanting natural forest frog fertilized eggs and tadpoles alone. In order to ensure the timely provision of excellent varieties with sufficient quantity, neat specifications, strong physique and rapid growth, the method of artificial propagation must be adopted to produce forest frog seedlings.

The artificial reproduction and production of forest frog has many advantages: first, the forest frog seedling is pure; second, it is convenient to raise and manage, and there can be a plan to produce seedlings according to needs and release them according to local conditions, so that the forest frog products are stable and high-yielding; third, it is self-breeding on the spot, avoiding long-distance transportation of seedlings and saving a lot of manpower and material resources; fourth, it can provide parent frogs and seedlings for local and surrounding breeding users to promote the development of forest frog breeding.

At present, the so-called artificial reproduction of forest frogs refers to artificial methods to promote female and male parent frogs to achieve sexual maturity under artificial breeding conditions, and can be in estrus, hug, mate and reproduce, so as to obtain fertilized eggs and give certain artificial conditions. so that love eggs develop and hatch. Therefore, the artificial reproduction of forest frog includes a series of processes, such as parent frog selection, transportation, intensive breeding, estrus, spawning, fertilized egg hatching and so on.

First, in addition to human technology and environmental factors, the quality of forest frog seedlings selected by parent frogs, the growth speed of young and adult frogs, and the spawning rate, fertilization rate, hatching rate and survival rate of young frogs in artificial reproduction, it also depends on the quality of parent frogs. Therefore, the choice of pro-frog is very important and must be taken seriously.

(1) selection criteria for pro-frog

1. Whether you choose to catch a natural wild parent frog, buy a parent frog from a farmer, or choose a parent frog from a self-raised backup parent frog, you should choose a frog aged 3 to 4 years old and over 6 years old, and it is not suitable to be a parent frog.

2. Individual specifications: female frogs are more than 7.5cm long and weigh more than 50g, while males are more than 6cm long and weigh more than 40g.

3. Physically strong, smooth and moist body surface, with many secretions; no disease, no injury, no deformity, complete appearance, exuberant vitality; individuals with good quality and typical forest frog characteristics.

(2) Identification and sex ratio of female and male frogs

1. It is very important to correctly identify the sex of forest frog between female and male frogs in the selection and actual production of parent frogs. Except for the identification of female and male according to the differences of internal structure and biological characteristics of forest frog, the difference between female and male appearance of forest frog mainly lies in the forelimb finger, those with tumor and strong protuberance (marriage pad) on the forelimb finger are male, and those without nodular protuberance are female.

2. The sex ratio of female and male frogs should be at 1:1, and the proportion of male frogs should not be too large. If it is too large, there will be a phenomenon of more males competing for one female, which will affect ovulation and fertilization. at the same time, it also increases the amount of feed and the competition of activity space, and increases the cost of breeding.

(3) choose the time to kiss the frog

The selection of parent frogs is best carried out in the early spring of each year, about a month before the forest frogs lay eggs. At this time, the forest frog has just finished hibernation, the weather is cool, the physiological activity of the forest frog is weak, the metabolism level is low, it is easy to transport, and the survival rate is high. And after the parent frogs are transported to the breeding ground, after short-term feeding and intensive breeding, they can enter into breeding.

The selection of parent frogs can also be carried out in late autumn when the forest frog is about to enter hibernation. However, after arriving at the breeding ground, it has to go through a long period of temporary rearing and a long hibernating period, which increases the workload of feeding and management, and during the overwintering period, the forest frog has low resistance to external environment and temperature and disease resistance, and there is often a risk of partial premature death.

From May to October when the temperature is high, it is not suitable for frog selection and long-distance transportation, during this period, the forest frog has vigorous physiological activity, high metabolic level, strong activity ability and easy to collide to death, and because of the high temperature, after long-distance transportation, it is easy to produce various diseases and lead to death. Second, parent frog transport because the young and adult forest frogs breathe with their lungs, they do not have to be soaked in water like fertilised eggs and tadpoles, as long as they can maintain a certain amount of humidity and ensure good breathability. it can be transported successfully by keeping a cool temperature.

(1) means of transport

Use wooden, iron, plastic, paper buckets or boxes, lined with smooth and soft plastic film or cloth, etc., the side of the barrel or box should drill multiple ventilation holes, pay attention to the lining do not plug the ventilation holes. The bottom of the bucket or box should be cushioned with thick sponge foam, water plants or cotton, cloth, etc., to maintain humidity and reduce vibration during transportation.

As the parent frog is large and active, in order to avoid collision and overlying death, it is best to divide the gear into several small chambers, each with 2 or 4 forest frogs. Young and adult frogs have strong bouncing ability. In order to prevent them from jumping out and escaping during transportation, the mouth should be sealed with wet cloth or close-eye mesh after packing.

(2) method of transport

The main results are as follows: 1. Stop eating 2-3 days before the parent frog box (bucket) is shipped, stop eating and do not feed, so as not to cause environmental pollution caused by excrement during transportation, and at the same time reduce its metabolic intensity and activity intensity. in order to facilitate transportation and improve the survival rate in transportation.

2. Transport density. Transport density can be loaded with 200,000 300 pieces per square meter of transportation equipment. The frog body should be washed clean before packing.

III. Intensive cultivation of parent frogs

Cultivating a certain number of high-quality parent frogs with good maturity, high fecundity and strong physique is the decisive material basis of artificial reproduction and the key to the success or failure of production in forest frog breeding. Therefore, the parent frogs must be reared scientifically and carefully, so as to achieve the goal of early maturity, high maturity rate and large number of eggs.

(1) Frog breeding ponds

Parent frog breeding ground is a collection of parent frog breeding, development, spawning breeding in one activity place, so it is also called spawning pool. The quality of the environment directly affects the gonadal development of the parent frog. Therefore, the area should not be too small, the area of each pool should be 20 to 30 square meters, one or more can be built according to the production scale. In order to facilitate feeding and management, to observe the spawning pairs and to collect fertilized egg clusters, a pool with a larger area can be built with a width of 4 to 5 meters and an arbitrary length, and its environment is as close to the natural state as possible. There should be land around or in the middle that occupies an area of water, and flowers or crops can be planted on the land as a shelter. The depth of the pool water does not have to be the same, but it can be deep or shallow, from 10 cm to 40 cm. Among them, about 10 centimeters of the "shallow water" area and 40 centimeters of the "deep water" area each account for half of the area. If the conditions are guaranteed, can maintain the freshness of the pool water and stable temperature, the pool water is about 10 centimeters of "shallow water" can also.

In the pond water, some aquatic plants such as paspalum, spring onion, Eichhornia crassipes and water hyacinth can be transplanted to meet the environmental conditions of spawning and reproduction of forest frog and purify the water quality at the same time. The pond should be surrounded by an anti-escape barrier to prevent the parent frog from escaping, with a total height of more than 1 meter, and the anti-escape barrier should be perpendicular to the ground or tilt slightly inward. The materials can be used locally, such as brick wall, earth wall, bamboo barrier, iron net, plastic sheet and so on.

(2) parent frog stocking

The stocking density of parent frogs was 2: 3 pairs per square meter, and the ratio of female to male frogs was 1:1. If the production scale is large and the number of parent frogs is large, the proportion of male frogs can be appropriately reduced, so as to prevent too many male frogs from fighting for female frogs and affecting their spawning pairs.

(3) cultivation and management

The nutritional status of parent frogs is not only directly related to the number of eggs and maturity, but also has a potential impact on the fertilization rate, hatching rate and even the survival rate of tadpoles and the growth of young frogs. Therefore, feeding parent frog feed requires rich and comprehensive nutrition, feed variety can not be single, as much as possible to feed some animal live bait. During the period of laying eggs, parent frogs have a high demand for nutrition and a large food intake, and their daily feeding should account for about 10% of their body weight, of which animal bait should account for 50% and 60% of the total feed. Feed twice a day, at dawn in the morning and before sunset in the afternoon, when the temperature is cooler.

During estrus and subsequent pairing of parent frogs, their food intake decreased significantly, and after spawning, they entered a state of reproductive dormancy and stopped feeding completely. During this period, bait should be appropriately reduced or stopped to feed for free. After the reproductive dormancy, the feeding of the parent frog returns to normal, and the quality and quantity of the feed still need to be guaranteed in time, so as to facilitate the recovery of the frog's physical fitness after giving birth and lay a good foundation for the next reproduction.

In the process of feeding and management of parent frogs, we should pay attention to adjust the water temperature, water quality and water level, timely and often inject and change the pool water, change the water at least twice a week, and keep the water temperature within the range of 18-24 degrees Celsius. It is necessary to do a good job in disease prevention and elimination at all times.

IV. Artificial induced labor

Under natural conditions, female and male frogs who reach sexual maturity begin to lay eggs in pairs as soon as the breeding season comes. However, due to the different maturity of parent frogs, coupled with the influence of climate and environmental factors, the time for parent frogs to lay eggs is first and later. Therefore, not only the breeding time is delayed for a long time, but also the specifications of the hatched tadpoles are uneven.

In order to make the parent frogs concentrate on laying eggs, shorten the breeding time and obtain neat seedlings, it is necessary to induce artificial spawning of the parent frogs.

(1) Identification of parent frog maturity

The mature female frog has a swollen and soft abdomen, has an obvious response to the call of the male frog in estrus, and responds with a low echo, is willing to approach and attach to the male frog, and likes to chase and play with it in groups on the surface of the water, not refusing the hug of the male frog. Such a female frog can lay eggs smoothly even if it is not artificially induced.

The female frogs with poor maturity had less abdominal enlargement and poor elasticity, were not interested in the male frogs, did not respond to the estrus calls of the male frogs, avoided the approach of the male frogs, preferred to move alone, and refused the hugs of the male frogs. For this part of the parent frog, artificial spawning must be given in order to achieve the goal of spawning at the same time as the mature parent frog.

(2) aphrodisiac drugs

At present, three kinds of aphrodisiac drugs are mainly used, such as chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogue (LRH-A) and forest frog pituitary. The first two drugs are sold commercially, while the pituitary gland of forest frog needs to be extracted and prepared by the user. The preparation method is as follows:

1. Take some adult forest frogs alive from the pituitary gland (not of the same species), cut them from the corners of both mouths to the back of the tympanic membrane with scissors, then cut the head horizontally behind the tympanic membrane, turn the skin of the oral maxilla forward to expose the skull, and use scissors to cut one knife obliquely from each side of the occipital foramen to the front of the eye; fold and lift the cut triangular bone pieces forward to expose the ventral side of the brain. The pituitary gland, a pink body buried in the lymphoid tissue, can be seen at the intersection of the optic nerve of the brain. The pituitary gland is removed from the connective tissue around the pituitary gland with sharp tweezers.

The pituitary gland taken out can be used freshly, and it can also be soaked in acetone or anhydrous alcohol for dehydration and degreasing, then dried and stored for spare, dry storage of the pituitary gland, which can be preserved for 1 to 2 years.

2. To prepare the medicine solution, the fresh pituitary gland or the dried pituitary gland was refined into powder or powder in the disinfected selenium, and then an appropriate amount of 0.65% normal saline was added to make the suspension, and the supernatant was used after precipitation.

(3) dose of aphrodisiac

Estrus drugs are mainly used in female frogs, because male frogs enter estrus about a week earlier than female frogs, and generally do not need to inject drugs to promote sperm production and maturation, but in order to promote their intensity of estrus, male frogs can also be injected with a dose equal to half of that of female frogs.

The above three drugs can be used alone or mixed, and the effect of general mixed use will be better.

When used alone, luteinizing hormone-releasing hormone analogue (LRH-A) was 30 μ g / kg body weight, chorionic gonadotropin (HCG) was 1200 international units / kg body weight, and the pituitary gland of forest frog was 1520 / kg body weight.

As the forest frog has a small body, there should not be too many kinds of aphrodisiac potions injected into the body, so the concentration of aphrodisiac drugs should be higher, and the volume of each parent frog injected into the body should be about 0.5 ml.

(4) injection method and effect time

The main results are as follows: 1. The injection method is gluteal muscle injection or abdominal subcutaneous injection. It usually only needs one injection. Buttock muscle injection, at a 45-degree angle, into the needle depth of about 1.5 cm. When abdominal hypodermic injection, pick up the belly with tweezers and pierce it horizontally with a needle depth of about 2 cm.

2. During the injection operation, it is best for two people to cooperate in the injection. One person holds the frog body with both hands, the other holds the syringe in the right hand, and the disinfection cotton ball gently reaches the injection site in the left hand. When the needle is withdrawn, the left hand gently kneads the pinhole with the cotton ball in the left hand to prevent the medicine from overflowing.

3. The effect time will put the parent frogs injected with aphrodisiac into the spawning pool according to the sex ratio at 1:1. Under the suitable water temperature, the effect time after induced labor is about 40-48 hours, and the female and male parent frogs begin to hug each other, and then ovulate, ovulate and complete the fertilization process. If there is still no hugging phenomenon for more than 48 hours, a second aphrodisiac is required, and the dosage of the second aphrodisiac is 1 big 2 of the first one.

4. To keep the environment quiet after injection, the ideal effect of artificial reproduction can be obtained by means of artificial spawning, natural spawning and fertilization. It is worth noting that when the parent frog is in estrus, the environment should be kept quiet, otherwise once stimulated by the outside world, the male frog will be stiff and it will take several minutes to recover. Therefore, artificial shock should be strictly prohibited so as not to affect the spawning and fertilization rate.

5. Artificial incubation

Under natural conditions, the hatching of fertilized eggs of wild forest frogs is often very low due to the harm of natural enemies and the influence of environment and climate. On the other hand, artificial hatching is carried out in a special hatching pool, which avoids the harm and influence of various unfavorable conditions and greatly improves the hatching rate.

(1) incubation pond

In order to hatch eggs laid in different periods in separate ponds and avoid uneven specifications of tadpoles caused by hatching eggs in different periods in the same pool, several more incubators can be built, depending on the number of parent frogs. The area does not need to be too large, generally 2 to 3 square meters, and the water depth is 20 to 30 centimeters. In order to ensure the content of dissolved oxygen in the water, the pool water should be able to maintain a certain exchange capacity, but the water flow should not be too large and the egg mass should not be impulsive. The water inlet and outlet should be covered with a sieve to prevent harmful insects, Cyclops, etc., from filling and fertilized eggs or tadpoles flowing out with the water.

If conditions permit, it is also possible not to build an incubation pool and hatch with a cage in a large water surface. The cage has an area of about 1 square meter and a depth of 40 centimeters, which is made of 40 mesh polyethylene mesh fixed on the frame. When hatching, the cage will be fixed on the surface of the water, the bottom of the cage into the water depth of about 15 cm, careful care and hatching.

(2) Hatching management

The main results are as follows: 1. After the fertilized eggs of the same batch of fertilized eggs are fully absorbed and expanded, they can be moved into the incubator to hatch. Eggs laid on the same day should be released in the same pool.

2. The hatching density can release 10-15 fertilized eggs, about 8000-15000 eggs per square meter.

3, water quality, water temperature control pool water should maintain micro-flow and exchange, water temperature should be kept at 18-24 degrees Celsius, do not exceed 27 degrees Celsius, otherwise embryos or tadpoles will be scalded to death. If it is still water hatching, replace the old water of 1x4 every 6 hours. The temperature difference between the newly injected water and the water in the original pool should not exceed 2 degrees Celsius.

4. The operation of egg delivery should be brisk when transferring fertilized eggs from the spawning pool to the hatching pond. Do not disperse the fertilized egg mass or reverse the top and bottom of the egg mass, otherwise it is easy to reduce the hatching rate or even lead to embryo death.

5. in order to find problems and sum up experiences and lessons, temperature, water depth, hatching time, number of hatched eggs, fertilization rate, hatching rate, hatching time and so on should be recorded carefully.

(3) embryonic development and metamorphosis

1. Embryonic development according to Ma Changfu's observation, the egg began to divide about 10 to 17 hours after fertilization (but at low water temperature, it began to divide at 2 to 3 days). On the second day of egg fertilization, that is, in the early stage of gastrum, the dorsal lip was initially formed and crescent-shaped grooves appeared. On the third day, entering the middle stage of the gastrula, the lateral lip appeared, the original mouth curled, and the dorsal lip and lateral lip formed a semicircular arc. On the 4th day, the inner lip of the original mouth rolled inward, the original mouth became smaller, blocked by yolk suppository, and transited to the late stage of gastrula. The nerve plate appeared on the 5th day, and then the edge of the nerve plate rose to form a fold, the center was concave inward, forming a nerve groove, the groove width was about 1 mm, and the embryonic body was 2.4 mm long. On the 5th day, the semi-nerve fold healed and formed a neural tube, the oral sucker appeared in a fissure shape, and the gills also appeared. From the 6th day to the 6th day and a half, the tail bud appeared, the length of the embryo body was 8mm, the length of the embryo body was 2.8 mm. On the 7th day, the outer Gill primordium was seen, the tail length was 1 × 3, and the embryo length was 6.5 mm. At this time, the embryo is detached from the egg gelatin membrane and adsorbed on objects such as the egg gel membrane or aquatic plants. On the 8th day, the outer Gill divides into short finger-shaped branches with a body length of 7.5 mm and a tail length of 1 stick 2. On the 9th day, the outer Gill was linear. The mouth of the tadpole was opened in about 10 days. On the 12th day of development, the body length is 11 mm, the tail length is 6 mm, the abdomen is short and round, the tail is flat and wide, and the digestive canal becomes curved. On the 13th day, the operculum formed, the outer Gill disappeared, the body length was 14 mm, the tail length was 8 mm, and the digestive canal was circuitous and curved. Since then, the embryonic development has been basically completed, and the next step is to enter the stage of development and perfection.

2. Metamorphosis develops from the fertilized egg to the 31st day, and the hindlimb begins to appear. The body length is 36mm, the tail length is 25mm, and the digestive canal is abnormally developed. On the 35th day, the forelimb appeared, the body was shortened to 34 mm, the tail length was 21 mm, the digestive duct began to contract and became smaller, and the intraoral horny plate disappeared. On the 37th day, the body length was shortened to 15 mm and the tail was shortened to 1 mm. At this point, the baby frog landed. On the 38th day, all the tails disappeared, and they were completely metamorphosed into young frogs.