Feed additive for fully artificially bred rana chensinensis and application of feed additive

Abstract

The invention relates to the technical field of artificial breeding, in particular to a feed additive for fully artificially bred rana chensinensis and application of the feed additive. The feed additive comprises dandelion and bacillus licheniformis, and the mass ratio of the dandelion to the bacillus licheniformis is (0.02-0.1): 1. The feed additive provided by the invention can effectively control the occurrence of digestive diseases of wood frog tadpoles, improve intestinal flora, promote weight gain and digestion of wood frog tadpoles, and significantly improve the metamorphosis effect of wood frog tadpoles and the survival rate of metamorphosed wood frog larvae.

Description

Technical Field

[0001] This invention relates to the field of artificial breeding technology, and more specifically, to a feed additive for fully artificially bred Northeast Forest Frog and its application. Background Technology

[0002] Artificial breeding techniques for amphibians have been promoted for many years and remain mature. While research on frog farming has been relatively limited in recent years, with increasing emphasis on food safety and sustainable resource utilization, research on fully artificial breeding techniques for frogs with high market value and medicinal / edible properties, such as the Northeast Forest Frog (Rana dybowskii), is gradually increasing. The Northeast Forest Frog (Rana dybowskii) is a tailless amphibian belonging to the Ranidae family and the Rana genus. As a frog with nutritional, medicinal, and meat-producing value, it is widely farmed, but most farmers concentrate on semi-wild environments, resulting in low recapture rates. Research on fully artificial breeding techniques for the Northeast Forest Frog is becoming increasingly mature and is currently being promoted; however, there is limited research on the scientific breeding of its tadpole stage. Tadpole breeding of the Northeast Forest Frog requires strict control over water quality and temperature; high-density farming can easily lead to disease and mass mortality. Furthermore, the feed formulations used by farmers are mostly based on the experience of farmers or feed manufacturers, lacking a unified standard for scientific feed formulation. The aquaculture industry urgently needs scientifically formulated feed to guide production, reduce breeding costs, and improve economic efficiency. The growth and digestive organ development of tadpoles and tadpoles of the Northeast Forest Frog directly affect their subsequent survival ability and reproductive success rate. Therefore, there is an urgent need for a feed specifically designed for the growth and development characteristics of the Northeast Forest Frog, which can increase stocking density while also preventing and treating diseases and reducing biological pollution.

[0003] Current research on amphibian feed mainly focuses on exploring feed substitutes from local resources and improving the nutritional composition of traditional feeds. Common research areas include optimizing the protein-carbohydrate ratio, adding vitamins and minerals, and utilizing alternative protein sources. While general-purpose tadpole feed research and preparation technologies are now widely applied in product manufacturing, research on different formulations required for different growth stages of Northeast Forest Frog tadpoles, as well as research on compound feeds containing traditional Chinese medicine, has not yet been conducted. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a feed additive for fully artificially bred Northeast Forest Frog and its application.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a feed additive for fully artificially bred Northeast Forest Frog, characterized in that it comprises dandelion and Bacillus licheniformis, wherein the mass ratio of dandelion to Bacillus licheniformis is (0.02~0.1):1.

[0006] In a preferred embodiment, the additive accounts for 10.2% to 11% of the total mass of the feed.

[0007] In a second aspect, the present invention provides a feed for the fully artificially bred Northeast Forest Frog, comprising a basic feed and the additives described in any of the above embodiments.

[0008] In the preferred embodiment, the basic feed, by weight, includes 3 parts corn flour, 2.5 parts soybean meal, 1.5 parts fish meal, 1.5 parts mealworm powder, 1 part wheat bran, 0.25 parts feed yeast powder, and 0.25 parts feed vitamin C.

[0009] Under the preferred method, the above-mentioned feed for fully artificially bred Northeast Forest Frogs is used for the tadpole stage of Northeast Forest Frogs.

[0010] A third aspect of the present invention provides the application of the feed additive for fully artificially bred Northeast Forest Frog described in any of the above embodiments in promoting the growth and development of Northeast Forest Frog.

[0011] In the preferred embodiment, the above applications include any one or more of the following: 1) Improve gut microbiota; 2) Promotes weight gain; 3) Promotes digestion; 4) Promotes tadpole metamorphosis; 5) Improve the survival rate of forest frog larvae after tadpole metamorphosis.

[0012] Beneficial effects of the present invention The feed additive provided by this invention can effectively control the occurrence of digestive diseases in forest frog tadpoles, improve intestinal flora, promote the weight gain and digestion of forest frog tadpoles, and significantly improve the metamorphosis effect of forest frog tadpoles and the survival rate of metamorphosed forest frog larvae. Attached Figure Description

[0013] Figure 1 This is a microscopic illustration of paraffin sections of the intestinal wall of tadpoles cultured for 30 days (left) and 50 days (right) in the CK group; Figure 2 This is a schematic diagram of paraffin sections of the intestinal wall of tadpoles cultured in the combination of dandelion and Bacillus licheniformis for 30 days (left) and 50 days (right). Figure 3 This is a microscopic illustration of a paraffin section of the stomach wall of 30-day-old tadpoles cultured in group T1. Figure 4 This is a microscopic image of a paraffin section of the stomach wall of 30-day-old tadpoles cultured in group T3. Figure 5 This is a microscopic illustration of a paraffin section of the stomach wall of 50-day-old tadpoles cultured in group T5. Figure 6 This is a microscopic illustration of a paraffin section of the stomach wall of 50-day-old tadpoles cultured from a combination of dandelion and Bacillus licheniformis. Figure 7 These are schematic diagrams (left) and (right) of the intestinal flora of 40-day-old tadpoles cultured in the CK group using Galanz staining biological microscopy and electron microscopy. Figure 8 These are schematic diagrams (left) and (right) of the intestinal flora of 40-day-old tadpoles cultured in group T5, obtained using Galanz staining biological microscopy. Figure 9 These are schematic diagrams (left) and (right) of the intestinal flora of 40-day-old tadpoles cultured in group T3, obtained using Galanz staining biological microscopy. Figure 10 These are schematic diagrams (left) and (right) of the intestinal flora of 40-day-old tadpoles cultured in group T7, obtained using Galanz staining biological microscopy. Figure 11 These are schematic diagrams (left) and (right) of the intestinal flora of 40-day-old tadpoles cultured in group T10, obtained using Galanz staining biological microscopy and electron microscopy. Figure 12 The images show schematic diagrams (left and right) of the intestinal flora of diseased tadpoles after treatment with Bacillus licheniformis using Galanz staining biological microscopy. Detailed Implementation

[0014] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0015] Unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise specified, the experimental methods used in the examples are conventional methods. Where specific conditions are not specified in the examples, they should be performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0016] Artificial breeding of frogs is now widely promoted, but different frog species have different nutritional requirements and rearing environments for their tadpoles. This invention focuses on the tadpoles of the Northeast Forest Frog, addressing the challenges of high-density breeding, high water quality requirements, and common diseases during their growth such as enteritis, red abdomen disease, and emphysema. It adds plant or microbial additives, including purslane, dandelion, wrinkled sorrel, and Bacillus licheniformis, to traditional pelleted feed to provide anti-inflammatory, antibacterial, and gut microbiota-regulating effects. The aim is to provide a scientific theoretical basis for the promotion of fully artificial and scientific breeding techniques for the Northeast Forest Frog.

[0017] 1. Materials and Methods 1.1 Experimental materials, reagents and instruments 1.1.1 Experimental Materials Plant materials: Fresh purslane, citronella, and dandelion collected from the mountains in the northeast of the region. After harvesting, they are washed with clean water to remove impurities and soil. After drying, they are crushed and set aside for later use.

[0018] Microbial preparation: Bacillus licheniformis, purchased from an online experimental materials procurement platform. Alternatively, Bacillus licheniformis obtained through ordinary channels with a viable count ≥100 billion / g can also be used to implement this invention and achieve the same technical effects.

[0019] Other feed ingredients: conventional feed ingredients such as soybean meal, corn flour, and fish meal, ensuring that they are of qualified quality and free from mold and contamination.

[0020] Preparation of artificially bred Northeast Forest Frog feed at different concentrations: All basic feed ingredients, purslane, sorrel, and dandelion were dried to ensure the moisture content of all raw materials was below 3%. All materials were pulverized through a 30-mesh sieve. 9%–11% of boiling water was added to the basic feed and stirred until coarse granules were formed. The mixture was then allowed to cool to below 38°C. Various feed additives were added according to the proportions in Table 1 and mixed thoroughly. A small pellet mill was used at approximately 8 kW power and a pressure of 0.3–0.4 MPa to press 3mm diameter pellets. The mill was stopped for 3 minutes every 10 minutes of operation to ensure the internal temperature of the pellets did not exceed 40°C, guaranteeing the survival rate of Bacillus. The finished feed was uniform in size and of moderate density, sinking in water and not completely dissolving within 5 minutes. The pelleted feed contained a certain amount of moisture; if not used within 3 days, it needed to be frozen or its moisture content reduced to an appropriate level using low-temperature drying equipment.

[0021] 1.1.2 Laboratory Animals Tadpoles hatched from eggs produced by the breeding frogs at a local artificial breeding base for Northeast Forest Frog were selected as experimental subjects. 10,000 healthy, disease-free, artificially bred Northeast Forest Frog larvae at 3 days old were selected.

[0022] 1.1.2 Experimental reagents and instruments Experimental equipment: electronic balance, pulverizer, breeding tank, microscope, dissection instruments, Olympus biological microscopy system, Hitachi S 3000N scanner electron microscope, processor, staining tank, coverslips, glass slides, forceps, warm water slide stage, blades, microtome, alcohol lamp, embedding cassette.

[0023] Experimental reagents: xylene, anhydrous ethanol, formaldehyde, paraffin, hematoxylin, eosin, crystal violet, iodine solution, 95% ethanol, Gram stain, differentiation solution (hydrochloric acid ethanol solution), mounting medium (neutral resin). 1.2 Experimental Grouping 3,300 artificially bred tadpoles of the Northeast Forest Frog with similar body size were randomly divided into 11 groups, with 3 replicates per group and 100 tadpoles per group.

[0024] The experimental groups and components are shown in Table 1 below, and are set according to gradient.

[0025] Table 1. Experimental groups and feed additive dosage for each group

[0026] 1.3 Feeding and Management During the experiment, the feeding and management were as follows: daily feed intake was 13% of body weight, fed twice daily; lighting conditions included natural light and darkness (approximately 14 hours of light: 10 hours of darkness). All feeding conditions remained consistent with the acclimatization period. During the acclimatization period, the feeding conditions were: water temperature 17–19℃, daily water exchange of 5% of total water volume, and dissolved oxygen content not less than 8%. Daily feed intake was 13%–15% of body weight, fed twice daily.

[0027] Every seven days, 20 tadpoles from each group are randomly selected for sampling, weighing, and length measurement to adjust the feeding amount. Since tadpoles undergo metamorphosis between 35 and 50 days of age, transitioning from aquatic to amphibious life, foam boards need to be added to the net cages during this period. This provides support for the tadpoles to meet the conditions for metamorphosis until their tails completely disappear, marking the completion of metamorphosis.

[0028] 1.4 Sample Collection During the experiment, the tadpoles in the net cage were counted and weighed every 7 days, and growth indicators were calculated.

[0029] During the experiment, every 9 days, the intestinal tissues of five juvenile or adult Northeast Forest Frogs were collected after fasting for 24 hours. Three 3cm segments of intestine were cut from the intestine of each frog. One segment was fixed with glutaraldehyde fixative for tissue sectioning, and the contents of the other segment were scraped for identification by scanning electron microscopy.

[0030] 1.5 Measurement Indicators Twenty tadpoles or frogs were randomly selected from each box and weighed. Body length, liver weight, visceral mass weight, and the number of surviving frogs were measured. The weight gain rate, condition factor, liver-to-body ratio, survival rate, and metamorphosis rate of the frog tadpoles were calculated. The calculation formulas are as follows: Weight gain rate (WGR) = (Final body mass - Initial body mass) / Initial body mass × 100% Condition factor (CF) = Final body weight / Body length 3 × 100% Viscerosomatic index (VSI) = visceral mass mass / final body weight mass × 100% Survival rate (SR) = (Number of fish remaining at the end / Number of fish at the beginning) × 100% In the above formula, the unit of mass is g and the unit of length is cm, and the same applies below.

[0031] Observe and record the incidence of diseases in forest frogs during the experiment, and compare the disease incidence rates between the compound feed and the control group. Dissect and examine the pathological characteristics of diseased and dead forest frog tadpoles to evaluate the effect of the compound feed on improving the disease resistance of forest frogs.

[0032] 1.6 Data Analysis The experimental results were processed using Excel 2010 and analyzed for variance using SPSS 20.0 statistical software.

[0033] 1.7 Microscopic identification and microscopic image analysis After each sampling, the tadpole intestinal tissue was longitudinally dissected under a stereomicroscope to expose the intestinal lumen. The intestinal contents were then spread into 90mm diameter disposable sterile culture dishes containing 10mL of lactose peptone medium. The inoculated culture dishes were placed in a 37℃ incubator for 24±2 hours, and a small number of mixed colonies were picked for Gram staining identification and scanning electron microscopy observation.

[0034] Gram staining identification of bacterial species Pick about 3mm 2 After the cultured mixed colonies were fixed with 95% ethanol for 10 minutes, crystal violet was added for staining for 1 minute, washed with water for 30 seconds, Gram's iodine solution was added for staining for 1 minute, 95% ethanol was added for decolorization for 25 seconds, tsar yellow was added for counterstaining for 1 minute, water was washed to remove excess color, and the colonies were dried before observation under a biological microscope.

[0035] Scanning electron microscopy of gut microbiota Pick about 3mm 2 The cultured mixed colonies were dispersed in 5 mL of PBS buffer, centrifuged and washed at 8000 rpm for 5 min, then washed three times with PBS solution, and fixed with 2.5% glutaraldehyde solution for 8 h. After washing with PBS solution and dehydrating with an ethanol gradient, 5 μL of the bacterial suspension was pipetted onto a silicon wafer and allowed to air dry. After gold ion sputtering coating, the cells were observed using a scanning electron microscope.

[0036] Microscopic observation of paraffin sections of digestive organs Tadpole intestinal tissue was fixed with 2.5% glutaraldehyde solution for 24 h after each sampling, and washed three times with PBS solution for 20 min each time. The tissue was then dehydrated using an ethanol gradient, cleared using a xylene gradient, and impregnated with a 9:1 paraffin-beeswax mixture. 12 μm thick paraffin sections were prepared using a paraffin microtome, dewaxed, rehydrated, stained with hematoxylin and eosin (HE), dehydrated, mounted, dried, and examined under a biological microscope.

[0037] 2. Experimental Results and Analysis 2.1 Effects of different additives on the growth performance of forest frog larvae Tadpoles aged 10 days (gill cover completion stage) to 40 days (abdominal contraction stage) were grouped and fed, and their final weight at 35 days of age, body length, survival rate, and internal organ weight were recorded. The data were then summarized and analyzed to form Table 2.

[0038] Table 2. Effects of different additives on the growth performance of 10-40 day old forest frog tadpoles

[0039] Note: Different lowercase letters after the data in the same column indicate significant differences (P<0.05), as in Table 3.

[0040] Data analysis revealed that, assuming tadpoles had relatively equal initial weights, the final average weights of tadpoles in the ten additive groups were significantly different from the control group (CK). Specifically, the purslane groups (T1-T2) consistently had lower final average weights than the control group (CK), and their weight gain rate was significantly lower. Comparing the final average weights of tadpoles in groups T1-T2 showed that higher purslane addition resulted in lower final weights. This indicates that purslane addition inhibits tadpole growth and is detrimental to weight gain. Except for groups T1-T2, the remaining eight additive groups consistently had significantly higher final average weights than the control group. Comparing the weight gain rate of tadpoles cultured in groups T5-T10 and the control group revealed that the addition of high-dose dandelion or high-dose Bacillus licheniformis promoted the weight gain of forest frog tadpoles, but the effect was not significant. The addition of low-dose dandelion or low-dose Bacillus licheniformis significantly promoted the weight gain of forest frog tadpoles, with weight gain rates of 288% and 283% in groups T5 and T7, respectively. Compared to the addition of only low-dose dandelion or very low-dose Bacillus licheniformis, the tadpoles cultured in the dandelion and Bacillus licheniformis combination group (T9-T10) showed significantly higher final average weight and growth rate, with weight gain rates of 292% and 296% in groups T5 and T7, respectively.

[0041] Regarding survival rates, except for the high-dose dandelion group (T6), where the survival rate of tadpoles was slightly lower than the control group, the survival rates of tadpoles in the other nine additive groups all improved. Among them, the survival rates of tadpoles in the *Rumex acetosa* group (T3-T4) and the low-dose dandelion group (T5) improved slightly, but the effect was not significant. However, the survival rates of *Bacillus licheniformis* groups (T7-T8) and the dandelion and *Bacillus licheniformis* combination groups (T9-T10) were significantly higher than other additive groups, indicating that the addition of *Bacillus licheniformis* is beneficial to improving the survival rate of *Frog.* When only *Bacillus licheniformis* was added to the feed, the survival rate of *Frog.* tadpoles showed a decreasing trend with increasing *Bacillus licheniformis* dosage; while when *Bacillus licheniformis* and dandelion were added to the feed, the survival rate of *Frog.* tadpoles showed an increasing trend with increasing *Bacillus licheniformis* dosage.

[0042] Regarding body condition, the tadpoles cultured in the *Bacillus licheniformis* group (T7-T8) showed a significant positive difference compared to other groups. Based on the body condition formula, it can be inferred that *Bacillus licheniformis* can promote the development of the visceral mass and cephalothorax, while inhibiting tail development. This finding has a favorable impact on tadpole survival. In comparing the viscera-to-body ratio, it was found that high-dose *Rumex rubra* added increased the viscera ratio. Dissection experiments revealed that the liver mass was larger than in other groups, suggesting that rhein in *Rumex rubra* may promote liver blood metabolism and stimulate liver development. Furthermore, the viscera-to-body ratio of tadpoles cultured in the dandelion and low-dose *Bacillus licheniformis* combination group (T9) and the dandelion and high-dose *Bacillus licheniformis* combination group (T10) were basically similar, and no liver enlargement was observed during dissection. Therefore, *Bacillus licheniformis* can promote the development of the digestive system.

[0043] A comprehensive analysis of the above experimental results shows that several additives can improve tadpole survival rates and inhibit disease occurrence to some extent. However, purslane is detrimental to tadpole weight gain, while wrinkled sorrel increases liver volume. Dandelion and Bacillus licheniformis both promote tadpole weight gain, but Bacillus licheniformis inhibits tail development to some extent. Adding a compound additive of 10% dandelion and 1% Bacillus licheniformis to the feed is most beneficial for the weight gain of forest frog tadpoles and can significantly improve their plumpness and survival rate.

[0044] 2.2 Effects of different added ingredients on metamorphosis of forest frog larvae After the tadpoles were raised to 35 days old, they gradually underwent metamorphosis from the forelimbs. The number of metamorphosed tadpoles in each group was recorded daily, and the data were summarized and analyzed to calculate the final metamorphosis rate. The weight of the metamorphosed tadpoles was also measured. The experimental breeding and observation continued for 7 days after the metamorphosis was completed and the caudal peduncle was completely absorbed. The feeding situation and survival rate after metamorphosis were recorded and finally summarized in Table 3.

[0045] Table 3. Effects of different added ingredients on metamorphosis of frog tadpoles

[0046] Analysis of Table 3 revealed that the control group fed the basal diet did not experience metamorphosis between 35 and 40 days of age, consistent with traditional frog farming practices. Metamorphosis in the control group mostly occurred between 55 and 70 days of age. Due to the relatively constant temperature in the laboratory environment, metamorphosis occurred 5 to 10 days earlier. Further analysis of the data in Table 3 showed that the final metamorphosis rate in all experimental groups was above 80%, indicating that most surviving tadpoles completed the metamorphosis process. This suggests that the feed provided all the necessary nutrients during the farming process. However, a large number of frogs died during the tail root stage before complete absorption of the caudal peduncle. The highest mortality rate, reaching 70%, was observed in the high-dose folic acid model group (T4). Preliminary analysis suggests that the high emodin content in the growing water stimulated the tadpoles, causing premature metamorphosis before their lungs were fully developed, leading to their inability to survive the terminal metamorphosis stage. Therefore, premature metamorphosis is not necessarily beneficial for tadpole survival.

[0047] After analysis and summarization, it was found that the high-dose purslane group (T2), the fruticosa group (T3~T4), and the dandelion group (T5~T6) could significantly promote the early metamorphosis of tadpoles. However, due to the early metamorphosis age, the weight of the tadpoles after metamorphosis in these groups was significantly lower than that of tadpoles that had metamorphosed at 40 to 50 days old. This phenomenon was not conducive to the survival of the tadpoles.

[0048] The metamorphosis time of the *Bacillus licheniformis* group (T7-T8) and the *Taraxacum mongolicum* and *Bacillus licheniformis* combination group (T9-T10) was concentrated between 40 and 50 days old, which is 5 to 7 days earlier than the control group and about 20 days earlier than the breeding base group. This time difference can effectively avoid the damage of high temperature to metamorphosing froglets. Comparing the weight of the metamorphosed froglets of the *Frog infestation* tadpoles in the T7-T10 groups and the control group, there was no significant difference in the weight of the metamorphosed froglets of the *Bacillus licheniformis* group and the control group; however, the weight of the metamorphosed froglets of the tadpoles in the *Taraxacum mongolicum* and *Bacillus licheniformis* combination group was significantly increased compared with the control group. The tadpoles in the *Taraxacum mongolicum* and low-dose *Bacillus licheniformis* combination group (T9) showed the best weight gain after metamorphosis, followed by the tadpoles in the *Taraxacum mongolicum* and high-dose *Bacillus licheniformis* combination group (T10). Therefore, it can be inferred that the compound additive of dandelion and Bacillus licheniformis can not only promote the early metamorphosis of young frogs, but also improve their survival rate after metamorphosis.

[0049] 2.3 Effects of different additives on the digestive system of forest frog larvae During the rearing process, we sampled and fixed the digestive organs of 30-day-old tadpoles and 50-day-old froglets, and then performed paraffin sectioning to compare the effects of several different additives on their digestive organs. During the comparison, we found... Figure 1 As shown, the intestinal wall of the 30-day-old tadpoles in the control group was thinner, the gastric wall epithelial cells were more prominent, and the gastric mucosa was less abundant. Figure 2 As shown, the intestinal tract of the dandelion combined with low-dose Bacillus licheniformis (T10) was thicker than that of the control group, the stomach wall was fuller and more robust, and the serous gland cells were thicker. Figure 3 The stomach wall of 30-day-old tadpoles in the low-dose purslane group showed a thicker gastric mucosa. Compared to tadpoles in the low-dose purslane group, the stomach wall of tadpoles in the low-dose folic acid model group at the same time was (…). Figure 4 The gastric mucosa is thin and adheres to the stomach wall, and the layers of stomach wall cells are not obvious. It can be seen that the rhein in the folic acid model caused a certain degree of stimulation to the stomach of the tadpoles. Figure 5 A transverse section of the intestinal wall of a 50-day-old frog in the low-dose dandelion group showed that the long villi were obvious and dense. Figure 6 The intestinal wall of 50-day-old froglets in the dandelion and low-dose Bacillus licheniformis compound group (T10) showed that the width of the long villi was wider than that in the dandelion group, and the long diameter of the long villi was longer than that in the dandelion-only group. This phenomenon indicates that the intestinal absorption capacity is better than that in the dandelion-only group.

[0050] 2.4 Effects of different added ingredients on changes in gut microbiota in forest frog larvae The results were consistent when observed using Galanz staining biological microscopes and electron microscopes. Figure 7The diagram shows the intestinal flora of the tadpoles in the control group, stained with Galanz stain under a biological microscope and an electron microscope. The intestinal flora contains a large number of Proteobacteria, which appear red under Galanz staining, and are suspected to be Escherichia coli, which can easily cause tadpole head red abdomen disease and enteritis. Figure 8 The diagram shows a schematic diagram of the gut microbiota of tadpoles in the low-dose dandelion group stained with Galanz stain and an electron microscope. The gut microbiota is relatively complex, including posterior wall bacteria and yeast. It is stained purple with Galanz stain and no red is seen. Most of the bacteria are positive, which is conducive to the growth and development of tadpoles. Figure 9 The images show schematic diagrams of the gut microbiota of tadpoles in group T3 stained under a biological microscope and an electron microscope. The number of bacteria in this group is relatively small, which indicates that the slow weight gain of the tadpoles in this group is related to the small number of gut microbiota. Figure 10 This is a schematic diagram of the intestinal flora of tadpoles stained with Galanz stain using a biological microscope and an electron microscope, showing the presence of numerous ungerminated posterior wall spores. The length of the Bacillus is relatively short. Figure 11 The combination of dandelion and Bacillus licheniformis has a shorter duration. Figure 11 Bacillus mesenteriae developed well and was the dominant bacterium. Figure 12 Tadpoles suffering from emphysema caused by enteritis were treated by transferring them to a water environment supplemented with Bacillus licheniformis at a concentration of 0.2 mg / L and feeding them with T10 feed. Fifteen days after treatment, observation showed that the emphysema in the tadpoles had largely healed, and their feeding and swimming had returned to normal. Microscopic observation of the intestinal flora after dissection revealed that Bacillus licheniformis proliferated extensively, invading the lesions occupied by Escherichia coli and inhibiting its growth, thus producing a beneficial effect on disease control in the tadpoles.

[0051] 3. Conclusion During the above experiments, the study found that the metamorphosis of forest frog tadpoles is closely related to the water quality of their living environment. A certain amount of harmful bacteria and parasites were detected in the water of the control group, severely affecting the growth and weight gain of the tadpoles. No parasites were found in the supplemented group. Purslane, Rumex rubra, and dandelion, three traditional Chinese medicinal plants, can effectively control digestive diseases in forest frog tadpoles. Although the addition of purslane and Rumex rubra can promote early metamorphosis in forest frog tadpoles, the addition of purslane significantly inhibits the weight gain of forest frog tadpoles and post-metamorphosed tadpoles, which is detrimental to the weight gain of forest frogs. The addition of Rumex rubra leads to an excessively large visceral-to-body ratio in forest frog tadpoles, significantly reducing the survival rate of post-metamorphosed tadpoles. Furthermore, high doses of purslane and Rumex rubra both affect the palatability of the tadpole feed. While low-dose dandelion or low-dose Bacillus licheniformis significantly promoted the weight gain of forest frog tadpoles in the early stages of the experiment, the weight of metamorphosed tadpoles in groups T5 and T7 did not significantly improve compared to the control group in the later stages. Compared to other additive groups, the dandelion and Bacillus licheniformis compound additive not only promoted forest frog metamorphosis and improved feed palatability but also significantly improved the weight of metamorphosed tadpoles, with a much higher survival rate than the control group. Among them, the 10% dandelion and 1% Bacillus licheniformis compound additive showed the best effects in improving intestinal flora, promoting weight gain, promoting digestion, and promoting metamorphosis, significantly improving the survival rate of metamorphosed forest frog larvae.

[0052] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A feed additive for fully artificially bred Northeast Forest Frogs, characterized in that, It includes dandelion and Bacillus licheniformis, wherein the mass ratio of dandelion to Bacillus licheniformis is (0.02~0.1):

1.

2. The feed additive for fully artificially bred Northeast Forest Frog as described in claim 1, characterized in that, The additives account for 10.2% to 12% of the total feed mass.

3. A feed for fully artificially bred Northeast Forest Frogs, characterized in that, Includes basic feed and the additives described in any one of claims 1 to 2.

4. The feed for fully artificially bred Northeast Forest Frogs according to claim 3, characterized in that, The basic feed, by weight, includes 3 parts corn flour, 2.5 parts soybean meal, 1.5 parts fish meal, 1.5 parts mealworm powder, 1 part wheat bran, 0.25 parts feed yeast powder, and 0.25 parts feed vitamin C.

5. The feed for fully artificially bred Northeast Forest Frogs according to claim 3, characterized in that, Used for the tadpole stage of the Northeast Forest Frog.

6. The application of the feed additive for fully artificially bred Northeast Forest Frog as described in any one of claims 1 to 2 in promoting the growth and development of Northeast Forest Frog.

7. The application according to claim 6, characterized in that, Includes one or more of the following: 1) Improve gut microbiota; 2) Promotes weight gain; 3) Promotes digestion; 4) Promotes tadpole metamorphosis; 5) Improve the survival rate of forest frog larvae after tadpole metamorphosis.

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