Detailed Explanation of Key Points of Artificial Breeding Technology in Forest Frog Farming

As the scale of forest frog farming continues to expand, the demand for forest frog fry grows ever greater, and relying solely on transplanting natural forest frog fertilized egg clusters and tadpoles can no longer meet the needs of farming. To ensure the timely supply of fine breeds that are sufficient in number, uniform in size, robust in constitution and fast-growing, artificial breeding methods must be adopted for forest frog fry production.

Artificial breeding production of forest frogs has many advantages: first, the forest frog fry are pure; second, feeding and management are convenient, allowing fry to be produced in a planned way according to need and stocked according to local conditions, so that forest frog products are stable and high-yielding; third, breeding and rearing on site avoids long-distance transport of fry and saves a great deal of manpower and material resources; fourth, it can provide parent frogs and fry to local and surrounding farming users, promoting the development of the forest frog farming industry.

The so-called artificial breeding of forest frogs currently refers to artificial methods that prompt female and male parent frogs, under artificial farming conditions, to reach sexual maturity and to be able to come into heat, amplexus, mate and reproduce, thereby obtaining fertilized eggs, and to provide certain artificial conditions so that the fertilized eggs develop and hatch into fry. Therefore, artificial breeding of forest frogs includes a whole series of processes such as parent frog selection, transport, intensive cultivation, induced spawning, egg laying and fertilized egg incubation.

I. Selection of Parent Frogs The quality of forest frog fry, the speed of growth of young and adult frogs, and the egg-laying rate, fertilization rate, hatching rate and survival rate of young frogs in artificial breeding all depend, apart from human technical and environmental factors, on the quality of the parent frogs. Therefore, the selection of parent frogs is crucial and must be taken seriously.

(I) Criteria for Selecting Parent Frogs

1. Age Whether selecting and catching wild parent frogs, purchasing parent frogs from farmers, or retaining parent frogs from self-reared reserve parent frogs, individuals of 3 to 4 years of age should be chosen; frogs over 6 years of age are not suitable as parent frogs.

2. Individual size Female frogs should have a body length of more than 7.5 cm and a body weight of more than 50 g; male frogs should have a body length of more than 6 cm and a body weight of more than 40 g.

3. Robust constitution The body surface should be smooth and moist with abundant secretions; free of disease, injury and deformity, with complete appearance and vigorous vitality; individuals of fine quality with typical forest frog characteristics.

(II) Identification of Female and Male Frogs and Sex Ratio

1. Identification of female and male frogs Correctly identifying the sex of forest frogs is very important in the selection of parent frogs and in actual production. In addition to identifying females and males based on differences in internal structure and biological characteristics, the external distinction between female and male forest frogs lies mainly in the front limbs and fingers: those with wart-like protuberances (nuptial pads) on the fingers of the front limbs are male, and those without wart-like protuberances are female.

2. Pairing sex ratio of female and male frogs The pairing sex ratio of female and male frogs is best at 1:1. The proportion of male frogs should not be too large; if too large, the phenomenon of multiple males competing for one female occurs, affecting amplexus, egg laying and fertilization, while also increasing the amount of feed supplied and competition for activity space, raising farming costs.

(III) Time for Selecting Parent Frogs

The work of selecting parent frogs is best carried out in early spring each year, about one month before forest frogs enter amplexus and lay eggs. At this time, forest frogs have just finished hibernation, the weather is cool, the physiological activity of the frogs is relatively weak and the metabolic level is relatively low, making transport convenient with a high survival rate. Moreover, after the parent frogs arrive at the farming site, a short period of rearing and intensive cultivation allows them to enter reproduction.

The work of selecting parent frogs can also be carried out in late autumn, just before the forest frogs enter hibernation. However, after arriving at the farming site, they must go through a relatively long period of temporary holding and a long overwintering hibernation period, increasing the workload of feeding and management; moreover, during overwintering, the frogs' resistance to changes in the external environment and temperature and to disease is relatively low, and there is often the danger that some may die prematurely.

In the hotter months from May to October, it is not suitable to select parent frogs or transport them over long distances. During this period, the physiological activity of forest frogs is relatively vigorous, the metabolic level is also relatively high and their activity capacity is strong, making them prone to colliding and dying; moreover, because of the high temperatures, long-distance transport easily produces various diseases that lead to death. II. Transport of Parent Frogs Since young and adult forest frogs breathe with lungs, during transport they do not need to be immersed in water as fertilized egg clusters and tadpoles are; as long as a certain humidity is maintained, good ventilation is ensured and a relatively cool temperature is kept, they can be transported successfully.

(I) Transport Tools

Wooden, iron, plastic or paper buckets or boxes can all be used, lined on the inside with smooth, soft plastic film or cloth, etc. The sides and top of the bucket or box must be drilled with multiple ventilation holes, and care must be taken that the lining does not block the ventilation holes. The bottom of the bucket or box must be padded with relatively thick, fully water-soaked sponge foam, aquatic plants, or cotton, cloth, etc., to maintain humidity and reduce vibration during transport.

Because parent frogs are relatively large and strong in activity, to avoid collision and crowding that could crush them to death, it is best to divide the container into several small compartments, with 2 to 4 forest frogs placed in each compartment. Young and adult frogs have strong jumping ability; to prevent them from jumping out and escaping during transport, after loading the box, the top opening should be sealed with a wet cloth or fine-mesh netting.

(II) Transport Methods

1. Fasting before transport Two to three days before the parent frogs are loaded into boxes (buckets) for transport, they must be fasted and no bait should be fed, so as to avoid excrement polluting the environment inside the transport container during the journey; at the same time, this reduces their metabolic intensity and activity intensity, which is beneficial for transport and raises the survival rate during transport.

2. Transport density The transport density can be 200 to 300 frogs per square meter of container area; before loading, the frogs' bodies should be washed clean.

III. Intensive Cultivation of Parent Frogs

Cultivating a certain number of high-quality parent frogs with good maturity, high fecundity and robust constitution is the decisive material foundation for artificial breeding in forest frog farming and the key to the success or failure of production. Therefore, parent frogs must be scientifically fed and carefully cultivated so as to achieve early maturity, a high maturity rate and a large number of eggs laid.

(I) Parent Frog Cultivation Pond

The parent frog cultivation site is an activity place that integrates parent frog rearing, development and amplexus, and egg laying and reproduction, so it is also called a spawning pond. The quality of its environment directly affects the gonadal development of the parent frogs. Therefore, the area should not be too small; the area of each pond is best 20 to 30 sq m, and one or several may be built depending on the scale of production. For ease of feeding and management, for ease of feeding and management, and for ease of observing amplexus and egg laying and collecting fertilized egg clusters, etc., a relatively large pond can be built as a narrow rectangular pond 4 to 5 m wide and of any length, with its environment as close to the natural state as possible. Land accounting for 1/3 of the water area should be left around or in the middle, and flowers, grasses or crops, etc., can be planted on the land as shelter. The depth of the pond water need not be uniform and can be deep or shallow, best from 10 cm to 40 cm. Among this, it is best for the “shallow water” area of about 10 cm and the “deep water” area of about 40 cm to each account for half. If conditions are guaranteed and the pond water can be kept fresh and the temperature stable, it is also acceptable for the pond water to be entirely “shallow water” of about 10 cm.

Some aquatic plants such as water barnyard grass, water onion, pondweed and water hyacinth can be transplanted into the pond water to satisfy the environmental conditions for forest frog egg laying and reproduction while also purifying the water quality. An escape-prevention barrier must be built around the pond to prevent parent frogs from escaping, with a height of more than 1 m; the barrier must be perpendicular to the ground or slightly inclined inward. Its materials can be obtained locally, and brick walls, earth walls, bamboo and wood barriers, iron netting, plastic sheeting, etc., can all be used.

(II) Stocking of Parent Frogs

The stocking density of parent frogs is 2 to 3 pairs per square meter, with a female-to-male ratio of 1:1. If the scale of production is relatively large and the number of parent frogs stocked is great, the proportion of male frogs can be appropriately reduced, to prevent excessive male frogs from fighting over females and affecting amplexus and egg laying.

(III) Cultivation Management

The nutritional status of parent frogs not only directly relates to the number of eggs carried 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, the feed fed to parent frogs must be rich and comprehensive in nutrition, the feed varieties must not be single, and as much animal-based live bait as possible should be fed. During the period when parent frogs are carrying and developing eggs, their nutritional requirements are relatively high and their food intake is large; the daily feeding amount should be about 10% of their body weight, and animal-based bait should account for 50% to 60% of the total feeding amount. Feed twice a day, in the early morning just after dawn and before sunset in the afternoon, when the temperature is relatively cool.

During the estrus period of parent frogs and the subsequent amplexus period, their food intake decreases markedly; after finishing egg laying, they enter a reproductive dormancy state and completely stop feeding. During this period, feeding should be appropriately reduced or stopped, to avoid wasting feed. After reproductive dormancy, the feeding of parent frogs returns to normal; at this time, feeding should be resumed promptly, and the quality and quantity of feed must still be guaranteed, to facilitate the recovery of the frogs' physical strength after spawning and lay a foundation for the next reproduction.

During the feeding and management of parent frogs, attention should be paid to regulating water temperature, water quality and water level, and pond water should be added and changed promptly and regularly, with water changed at least twice a week; the water temperature should be kept within the range of 18 to 24 degrees Celsius and must not fluctuate between hot and cold. Disease prevention and pest elimination, etc., must be done at all times.

IV. Artificial Induction of Spawning

Under natural conditions, once the breeding season arrives, sexually mature female and male frogs begin amplexus, egg laying and reproduction. However, because the maturity of parent frogs varies and because of the influence of climate and environmental factors, etc., the timing of amplexus and egg laying differs from one individual to another. Therefore, not only is the breeding period greatly prolonged, but the tadpoles hatched are also uneven in size.

In order to enable parent frogs to concentrate on amplexus and egg laying, shorten the breeding time and obtain fry of uniform size, it is necessary to carry out artificial induction of spawning in parent frogs.

(I) Identification of Parent Frog Maturity

A well-matured female frog has a distended, soft abdomen, reacts obviously to the estrus calls of male frogs and responds with low answering calls, is willing to approach and attach to male frogs, likes to gather and chase and play with them on the water surface, and does not refuse the male frog's embrace. Such a female frog can lay eggs smoothly even without artificial induction of spawning.

A female frog of poorer maturity has a lesser degree of abdominal distension and poor elasticity, is uninterested in male frogs, does not react to the estrus calls of male frogs, avoids the approach of male frogs and likes to move alone, and refuses the embrace of male frogs. For these parent frogs, artificial induction of spawning must be applied to achieve the goal of laying eggs at the same time as well-matured parent frogs.

(II) Spawning-Inducing Drugs

The spawning-inducing drugs currently used mainly include three kinds: human chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogue (LRH-A) and forest frog pituitary, etc. The first two drugs are sold commercially, while forest frog pituitary must be extracted and prepared by the user; the preparation method is:

1. Extracting the pituitary Take several live adult forest frogs (other species are also acceptable), use scissors to cut open from the corners of the mouth on both sides to behind the tympanic membrane, then cut off the head horizontally behind the tympanic membrane; turn the skin of the upper jaw of the oral cavity forward to expose the skull, and use scissors to make one oblique cut from each side's foramen magnum toward the front of the eye; fold and lift the resulting triangular bone flap forward to expose the ventral surface of the brain; at the optic chiasm of the brain, a small pink body buried in lymphatic tissue can be seen - the pituitary; use fine tweezers to separate the connective tissue around the pituitary and take out the pituitary.

The extracted pituitary can be used fresh, or after being soaked in acetone or anhydrous alcohol for dehydration and degreasing, it can be dried and stored for later use; pituitaries stored dry can be kept for 1 to 2 years.

2. Preparing the medicinal solution Grind fresh pituitaries or pituitaries preserved dry into a fine powder or powder in a disinfected mortar, then add an appropriate amount of 0.65% physiological saline to make a suspension, and after settling, take the supernatant liquid for use.

(III) Dosage of Spawning-Inducing Drugs

Spawning-inducing drugs are mainly used on female frogs, because male frogs enter the estrus period about one week earlier than female frogs and generally do not need drug injection to promote the production and maturation of sperm, but in order to promote the intensity of their estrus, male frogs may also be injected as appropriate with a dose equivalent to half that for female frogs.

The above three drugs can be used alone or in combination; generally, combined use gives better results.

When used alone, luteinizing hormone-releasing hormone analogue (LRH-A) is 30 micrograms/kg body weight; human chorionic gonadotropin (HCG) is 1200 international units/kg body weight; forest frog pituitary is 15 to 20 per kg body weight.

Because the forest frog's body is relatively small, the volume of spawning-inducing medicinal solution injected into the body must not be too much, so the concentration of the spawning-inducing drug should be prepared somewhat higher, and the volume of spawning-inducing medicinal solution injected into each parent frog is best about 0.5 mL.

(IV) Injection Method and Effect Time

1. Injection method Use gluteal muscle injection or abdominal subcutaneous injection. Generally only one injection is needed. For gluteal muscle injection, insert at a 45-degree angle, with a needle insertion depth of about 1.5 cm. For abdominal subcutaneous injection, use tweezers to lift the belly skin and insert horizontally, with a needle insertion depth of about 2 cm.

2. Injection procedure For spawning-inducing injection, it is best for two people to cooperate: one person holds the frog's body with both hands, and one person holds the syringe in the right hand while using the left hand to lightly press a sterilized cotton ball against the injection site; when withdrawing the needle, the left hand gently presses and massages the needle hole with the cotton ball to prevent the medicinal solution from overflowing.

3. Effect time Place the parent frogs injected with spawning-inducing drugs into the spawning pond at a sex ratio of 1:1. At a suitable water temperature, the effect time after induced spawning is about 40 to 48 hours, when female and male parent frogs begin amplexus, then release eggs and sperm to complete the fertilization process. If after more than 48 hours there is still no amplexus, a second induced spawning is needed, and the dosage for the second induced spawning is 1/2 that of the first.

4. Keep the environment quiet after injection Using the method of artificial induced spawning and natural spawning and fertilization can achieve ideal artificial breeding results. It is worth noting that when parent frogs are in estrus and amplexus, the environment should be kept quiet; otherwise, once stimulated by the outside world, male frogs will become rigid and need several minutes to recover. Therefore, human disturbance must be strictly prohibited, so as not to affect egg laying and the fertilization rate.

V. Artificial Incubation

Under natural conditions, the fertilized eggs of wild forest frogs often have a very low hatching rate due to the harm of natural enemies and the influence of environmental and climatic conditions. Artificial incubation, however, is carried out in dedicated incubation ponds, avoiding the harm and influence of various adverse conditions and greatly raising the hatching rate.

(I) Incubation Pond

In order to incubate eggs laid at different times in separate ponds and avoid the phenomenon of uneven tadpole size caused by incubating eggs from different periods in the same pond, several incubation ponds can be built, with the number depending on the number of parent frogs. The area need not be too large; generally 2 to 3 sq m is sufficient, with a water depth of 20 to 30 cm. To ensure the dissolved oxygen content in the water, the pond water should be able to maintain a certain exchange volume, but the water flow must not be too strong and must not disturb the egg clusters. Both the water inlet and outlet must be covered with sieve silk, to prevent harmful insects, cyclops, etc., from entering and fertilized eggs or tadpoles from flowing out with the water.

If conditions permit, incubation ponds need not be built, and net cages can be used for incubation in large water bodies. The net cage area is about 1 sq m, the cage depth is 40 cm, and it is made by fixing 40-mesh polyethylene netting onto a frame. During incubation, the net cage fixing frame is set up on the water surface, with the bottom of the net cage submerged to a depth of about 15 cm, and careful watching over the incubation is all that is needed.

(II) Incubation Management

1. Place fertilized eggs of the same batch After the fertilized egg clusters have fully absorbed water and swollen, they can be moved into the incubation pond for incubation. In the same pond, eggs laid on the same day must be placed.

2. Incubation density Per square meter, 10 to 15 fertilized egg clusters can be placed, about 8000 to 15000 eggs.

3. Water quality and water temperature control The pond water should maintain slight flow and exchange, and the water temperature should be kept at 18 to 24 degrees Celsius; it must never exceed 27 degrees Celsius, otherwise the embryos or tadpoles will be scalded to death. If incubating in still water, 1/4 of the old water should be replaced every 6 hours, and the temperature difference between the newly added water and the original pond water must not exceed 2 degrees Celsius.

4. Egg transfer operations should be light and quick When moving fertilized egg clusters from the spawning pond to the incubation pond, the operation should be light and quick; the fertilized egg clusters must not be broken apart or turned upside down, otherwise the hatching rate is easily reduced and the embryos may even die.

5. Keep good incubation records Carefully record temperature, water depth, time of placement in incubation, number of eggs placed in incubation, fertilization rate, hatching rate, hatching time, etc., so that problems can be found and dealt with promptly and experience and lessons can be summarized.

(III) Embryonic Development and Metamorphosis Process

1. Embryonic development According to the observations of Ma Changfu et al., about 10 to 17 hours after fertilization the egg begins to divide (but under low water temperature conditions, division begins only after 2 to 3 days); on the second day after fertilization, it enters the early gastrula stage, first forming the dorsal lip and appearing as a crescent-shaped small groove. On the third day it enters the mid-gastrula stage, the lateral lips appear, the blastopore curls inward, and the dorsal lip and lateral lips join to form a semicircular arc. On the fourth day the inner lip of the blastopore curls inward, the blastopore becomes smaller and is blocked by the yolk plug, transitioning to the late gastrula stage. On the fifth day the neural plate appears, after which the edges of the neural plate rise to form folds, and the center sinks inward to form the neural groove, which is about 1 mm wide, with an embryo body length of 2.4 mm. On the fifth and a half day the neural folds fuse to form the neural tube, the oral sucker appears in the shape of a slit, and gills also appear. From the sixth day to the sixth and a half day, the tail bud appears, with a length of 1/8 to 1/9 of the embryo body length, and the embryo body length is 2.8 mm. On the seventh day the external gill primordia are visible, the tail length is 1/3 of the total length, and the embryo length is 6.5 mm. At this time the embryo leaves the egg jelly membrane and attaches to the egg jelly membrane or objects such as aquatic plants. On the eighth day the external gills divide into short finger-like branches, the body length is 7.5 mm, and the tail length is 1/2 of the body length. On the ninth day the external gills become filamentous. At about 10 days the tadpole's mouth has opened. By the twelfth 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 tract becomes curved. On the thirteenth day the operculum forms, the external gills disappear, the body length is 14 mm, the tail length is 8 mm, and the digestive tract is tortuous and curved. From this point, embryonic development is basically complete, and the next step enters the stage of developmental refinement.

2. Metamorphosis process From the thirty-first day of development from the fertilized egg, the hind limbs begin to appear, the body length is 36 mm, the tail length is 25 mm, and the digestive tract is unusually developed. On the thirty-fifth day the forelimbs appear, the body shortens to 34 mm, the tail length is 21 mm, the digestive tract begins to contract and become smaller, and the keratinous plates in the mouth disappear. On the thirty-seventh day, the body length shortens to 15 mm and the tail shortens to 1 mm. At this time the young frog comes onto land. On the thirty-eighth day, the tail completely disappears, and to this point it has completely metamorphosed into a young frog.