A method for breeding of gold pompano against heat stress

Abstract

The application provides a kind of gold pompano heat stress resistance cultivation method, using domestication pool I, II, III, according to juvenile stage, juvenile stage, adult fish stage, gold pompano is domesticated, in juvenile stage, using bait domestication, through the specific bait formula in nutrition or pharmacological means, the fat utilization ability of fish body can improve the heat stress tolerance of fish, temperature domestication is carried out in juvenile stage, four-stage temperature domestication is adopted, the heat tolerance of fish body is gradually trained, the defense ability of non-specific immunity of fish body is improved, sound wave domestication is carried out in adult fish stage, the sound information is transmitted to the motor-related neuron in hindbrain, so as to promote the tail movement of itself, improve the immune function of fish body, the application domesticates the feeding, enemy, circadian rhythm regulation ability of fish body, improves the physical fitness of fish body to enhance the heat reaction ability, reduces the probability of heat reaction, at the same time, can better, faster adapt to harsh breeding environment, the survival rate reaches 98.8% after stress test, and the weight loss rate is-0.02%.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fish culture, in particular to a method for culturing Trachinotus ovatus with heat stress resistance. BACKGROUND

[0002] Trachinotus ovatus, also known as oval jack, is one of the precious marine economic fish in the south coast of China. It is rich in protein, amino acids, unsaturated fatty acids, DHA and other nutrients, and has high nutritional value, high food value and high economic value. Because the fish meat is tender, delicious, high in nutritional value and free of small intermuscular spines, it is deeply loved by consumers.

[0003] As poikilothermic vertebrates, fish cannot maintain a relatively stable body temperature like mammals, and the body temperature of fish will change with the change of water temperature. Due to the unstable body temperature, fish are more susceptible to water temperature than mammals. Temperature can comprehensively affect various life activities of fish, including development, growth, feeding, digestion and absorption, movement, reproduction, immunity and even cause fish death. However, the environmental temperature in nature is always affected by various factors and changes constantly, the most common of which is the alternation of day and night and the change of seasons, which can cause the temperature to change periodically. Climate change and human activities can also change the temperature of the environment such as air and water. Although water has a large specific heat capacity, its temperature fluctuation is smaller than that of air, but it is always in dynamic fluctuation. The gradual warming of global climate leads to the rising trend of water temperature, and the heat stress faced by aquatic animals has attracted widespread attention. If the high temperature lasts for a long time, it will not only inhibit the growth of fish and cause the body to weaken, but also affect the growth and reproduction of some fish, and even cause the death of fish. Therefore, the adverse effects of high temperature on fish are extremely serious.

[0004] Stress is the sum of nonspecific responses of the body to various external or internal stimuli. For fish, when the water temperature exceeds the optimum water temperature of the fish body, the fish body will produce a stress response to protect its normal metabolic function, which is a protective response, but the function of the nonspecific immune defense system of the body will be inhibited if the fish body is continuously in stress. The normal blood indicators of animals can reflect the normal physiological state of animals. Because blood is closely related to the metabolism, nutritional status and diseases of the body, when the fish body is stimulated by external stimuli and physiological or pathological changes occur, it will be reflected in the blood indicators. SUMMARY

[0005] In view of this, the present application provides a method for culturing Trachinotus ovatus with heat stress resistance to solve the above problems.

[0006] The technical scheme of the present application is implemented as follows: a gold pompano heat stress resistance breeding method: gold pompano fry is put into domestication ponds I, II and III, and the gold pompano is domesticated according to the juvenile stage, the young fish stage and the adult fish stage, and the method specifically comprises the following steps:

[0007] S1, food induction domestication: healthy fry is selected and put into domestication pond I, the fry is put at a density of 150-180 thousand tails / m 3 , artificial compound feed is fed, the feed is fed 3-5 times a day, the feed amount is 2-5% of the weight of the fry, feeding is continuously performed for 5 days and then stopped for 2 days, and the feeding is sequentially and periodically performed until the young fish stage;

[0008] S2, temperature domestication: the young fish is put into domestication pond II, the young fish is put at a density of 220-280 tails / m 3 , and four-stage temperature domestication is performed, the first stage is set to a water temperature of 22-25 DEG C, water flow stimulation is performed for 15-20 min, the second stage is set to a water temperature of 26-30 DEG C, water flow stimulation is performed for 20-25 min, the third stage is set to a water temperature of 31-35 DEG C, water flow stimulation is performed for 11-14 min, the fourth stage is set to a water temperature of 36-40 DEG C, and water flow stimulation is performed for 5-10 min, and the continuous domestication is performed for 2 weeks;

[0009] S3, sound wave domestication: after the temperature domestication, the young fish is transported to domestication pond III when the young fish grows to the adult fish stage, the young fish is put at a density of 100-120 tails / m 3 , sound wave driving is performed on the fish group every 8-14 h every day, each driving lasts for 150-300 s, artificial compound feed is uniformly fed at the same time, and the period lasts for 18-22 days;

[0010] S4, daily management: the domestication pond is patrolled in the morning and evening, and residual feed is cleaned in time, water is changed every 3-5 days, and the water changing amount is 50-80%.

[0011] Further, the water quality management conditions in the domestication ponds I, II and III are as follows: the pH value is 7.6-8.2, the dissolved oxygen is greater than 5 mg / L, the ammonia nitrogen is less than 0.2 mg / L, the salinity is 22-36, the transparency is 70-90 cm, and the nitrite content is 0.021-0.058 mg / L.

[0012] Further, in S2, temperature domestication is performed twice every other day in the morning from 8:00 to 9:00 and in the afternoon from 3:00 to 4:00, and the water flow rate is controlled to be 0.8-1.5 m 3 / h.

[0013] Further, in S2, after each temperature domestication, artificial compound feed is fed, and the feed amount is 5-10% of the weight of the young fish.

[0014] Further, in S3, the feed amount is 6-18% of the weight of the adult fish.

[0015] Further, the S1-S3 domestication is simultaneously managed by red light illumination, and the illumination is started 0.5-1h before each time of feeding the artificial compound bait, the illumination intensity is 600-1000lx, and the illumination time is 1-3h / d.

[0016] Further, the artificial compound bait comprises the following raw materials in parts by weight: corn flour 20-50 parts, fish bone powder 12-33 parts, spirulina powder 13-22 parts, yao column powder 5-8 parts, grape seed powder 8-15 parts, red skin garlic 2-6 parts, taurine 1.2-8.5 parts, and additives 2-4 parts.

[0017] Further, the artificial compound bait comprises the following raw materials in parts by weight: corn flour 33 parts, fish bone powder 25 parts, spirulina powder 18 parts, yao column powder 7 parts, grape seed powder 12 parts, red skin garlic 4 parts, taurine 6.2 parts, and additives 3 parts.

[0018] Further, the additives are composed of luteolin, limonin, gastrodin polysaccharide and gingerol in a mass ratio of (6-10):(3-8):(12-25):(0.3-0.9).

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application domesticates the golden pompano in the domestication pools I, II and III according to the juvenile fish period, the young fish period and the adult fish period, uses the bait domestication in the juvenile fish period, improves the immune ability and the oxidation stress resistance of the fish body, improves the fat utilization ability of the fish body through the nutrition or pharmacological means in the specific bait formula, improves the heat stress resistance of the fish, scientifically selects the raw materials in the bait and reasonably matches the ratio, reduces the 5-hydroxyproline content through the glutathione content in the liver, reduces the oxidation stress of the liver, removes the free radicals, improves the antioxidant ability, promotes the anti-heat stress ability, trains the heat resistance of the fish body gradually through the temperature domestication in the young fish period, improves the defense ability of the non-specific immunity of the fish body, and promotes the immune function of the fish body, maintains the blood balance, and improves the adaptation ability of the golden pompano to the environment through the sound wave domestication in the adult fish period.

[0021] The application domesticates the feeding, enemy resistance and diurnal rhythm regulation ability of fish, turns on the light before putting the artificial mixed bait, causes cell apoptosis through the mitochondrial pathway to maintain the stability of the tissue in response to heat stress, thereby improving the constitution of the fish to enhance the heat resistance ability, reducing the probability of heat resistance, and better and faster adapting to harsh breeding environment, and laying a foundation for breeding high-temperature-resistant golden pompano varieties in the future. DETAILED DESCRIPTION

[0022] In order to better understand the technical content of the application, the following specific examples are provided to further illustrate the application.

[0023] The experimental methods used in the embodiments of the application are conventional methods unless otherwise specified.

[0024] The materials, reagents and the like used in the embodiments of the application can be obtained from commercial channels unless otherwise specified.

[0025] Example 1

[0026] A heat stress resistance breeding method of golden pompano: golden pompano fry is put into domestication pools I, II and III, and the water quality management conditions in the domestication pools I, II and III are as follows: pH value 7.6, dissolved oxygen > 5 mg / L, ammonia nitrogen < 0.2 mg / L, salinity 22, transparency 70 cm, nitrite content 0.021 mg / L, and the golden pompano is domesticated according to the juvenile stage, the young fish stage and the adult fish stage, and red light illumination management is carried out at the same time, and the light is turned on 0.5 h before each time the artificial mixed bait is put, the light intensity is 600 lx, the illumination time is 1 h / d, and the method specifically comprises the following steps:

[0027] S1, feeding domestication: healthy fry is selected and put into the domestication pool I, the fry is put at a density of 150,000 tails / m 3 , artificial mixed bait is fed, the fry is fed 3 times a day, the feeding amount is 2% of the weight of the fry, feeding is stopped for 2 days after continuous feeding for 5 days, and periodic feeding is carried out in turn until the young fish stage;

[0028] S2, temperature domestication: the above young fish is put into the domestication pool II, the young fish is put at a density of 220 tails / m 3 , temperature domestication is carried out twice at 8:00-9:00 in the morning and 3:00-4:00 in the afternoon every other day, four-stage temperature is used for domestication, the first stage is set to 22℃, water flow stimulation is 15 min, the second stage is set to 26℃, water flow stimulation is 20 min, the third stage is set to 31℃, water flow stimulation is 11 min, and the fourth stage is set to 36℃, water flow stimulation is 5-10 min, the water flow rate is controlled to be 0.8 m 3 / h, and continuous domestication is carried out for 2 weeks;

[0029] S3, sound wave domestication: after temperature domestication, the juvenile fish is grown into adult fish, and is transported to a domestication pool III, and the feeding density is 100 tails / m 3 The sound wave driving is performed on the fish group every 8 hours per day, the sound wave frequency is 900 Hz, each driving lasts for 150 seconds, the sound wave is emitted while the artificial bait is uniformly fed, the feeding amount is 6% of the weight of the adult fish, and the period lasts for 18 days.

[0030] S4, daily management: early and late inspection of the domestication pool, timely cleaning of residual bait, water replacement every 3 days, and the water replacement amount is 50-80%.

[0031] The artificial bait includes the following raw materials in parts by weight: 20 parts of corn flour, 12 parts of fish bone powder, 13 parts of spirulina powder, 5 parts of conch powder, 8 parts of grape seed powder, 2 parts of red-skinned garlic, 1.2 parts of taurine, and 2 parts of additives, and the additives are composed of luteolin, limonin, gastrodin polysaccharide and gingerol at a mass ratio of 6:3:12:0.3.

[0032] Example 2

[0033] A goldfish anti-heat stress breeding method: goldfish fry is put into domestication pools I, II and III, and the water quality management conditions of the domestication pools I, II and III are as follows: pH value 8.2, dissolved oxygen > 5 mg / L, ammonia nitrogen < 0.2 mg / L, salinity 36, transparency 90 cm, nitrite content 0.058 mg / L, and the goldfish is domesticated according to the juvenile stage, the juvenile stage and the adult stage, and red light illumination management is performed at the same time, the red light is turned on 1 hour before each time of feeding the artificial bait, the illumination intensity is 1000 lx, and the illumination time is 3 hours per day, and the method specifically comprises the following steps:

[0034] S1, feeding domestication: healthy fry is selected and put into a domestication pool I, and the fry feeding density is 180,000 tails / m 3 The artificial bait is fed, the feeding amount is 5% of the weight of the fry, the feeding is stopped for 2 days after 5 days of continuous feeding, and the feeding is sequentially and periodically performed until the juvenile stage;

[0035] S2, temperature domestication: the above juvenile fish is put into a domestication pool II, and the juvenile fish feeding density is 220-280 tails / m 3 Temperature domestication is performed twice every other day from 8:00 to 9:00 in the morning and from 3:00 to 4:00 in the afternoon, four-stage temperature is adopted for domestication, the first stage is set to 25℃, water flow stimulation is performed for 20 minutes, the second stage is set to 30℃, water flow stimulation is performed for 25 minutes, the third stage is set to 35℃, water flow stimulation is performed for 14 minutes, and the fourth stage is set to 40℃, water flow stimulation is performed for 10 minutes, the water flow speed is controlled to be 1.5 m 3 / h, and the continuous domestication lasts for 2 weeks;

[0036] S3, sound wave domestication: after temperature domestication, the juvenile fish is grown into adult fish, and is transported to a domestication pool III, and the feeding density is 120 tails / m 3 , the sound wave frequency is 1100 Hz, each driving lasts for 300 seconds, and the artificial bait is uniformly fed at the same time, the feeding amount is 18% of the weight of the adult fish, and the period lasts for 22 days;

[0037] S4, daily management: early and late inspection of the domestication pool, timely cleaning of residual bait, water replacement for 5 days, and the water replacement amount is 50-80%.

[0038] The artificial bait includes the following raw materials in parts by weight: corn flour 50 parts, fish bone powder 33 parts, spirulina powder 22 parts, conch powder 8 parts, grape seed powder 15 parts, red-skinned garlic 6 parts, taurine 8.5 parts, and additives 4 parts, and the additives are composed of luteolin, limonin, gastrodin polysaccharide and gingerol in a mass ratio of 10:8:25:0.9.

[0039] Example 3

[0040] A goldfish anti-heat stress breeding method: goldfish fry is put into domestication pools I, II and III, and the water quality management conditions of the domestication pools I, II and III are as follows: pH value 8, dissolved oxygen > 5 mg / L, ammonia nitrogen < 0.2 mg / L, salinity 30, transparency 80 cm, nitrite content 0.035 mg / L, and the goldfish is domesticated according to the juvenile stage, the juvenile stage and the adult stage, and red light illumination management is carried out at the same time, the red light is turned on 0.8 hours before each time of feeding the artificial bait, the illumination intensity is 800 lx, and the illumination time is 2 hours per day, and the method specifically comprises the following steps:

[0041] S1, feeding domestication: healthy fry is selected and put into a domestication pool I, and the fry feeding density is 160,000 tails / m 3 , the artificial bait is fed, the feeding amount is 4% of the weight of the fry, the feeding is stopped for 2 days after continuous feeding for 5 days, and the feeding is sequentially and periodically carried out until the juvenile stage;

[0042] S2, temperature domestication: the above juvenile fish is put into a domestication pool II, and the juvenile fish feeding density is 250 tails / m 3 , temperature domestication is carried out twice every other day from 8:00 to 9:00 in the morning and from 3:00 to 4:00 in the afternoon, four-stage temperature is used for domestication, the first stage is set to 23℃, water flow stimulation is 18 minutes, the second stage is set to 28℃, water flow stimulation is 23 minutes, the third stage is set to 33℃, water flow stimulation is 13 minutes, and the fourth stage is set to 38℃, water flow stimulation is 8 minutes, the water flow speed is controlled to be 1.2 m 3 / h, and the continuous domestication lasts for 2 weeks;

[0043] S3, sound wave domestication: after temperature domestication, the young fish is grown into adult fish, and is transported to the domestication pool III, with a density of 110 tails / m 3 The sound wave is used to drive the fish every 11 hours per day, with a frequency of 1000 Hz, and each driving lasts for 230 seconds. The artificial bait is fed at the same time, with a feeding amount of 12% of the weight of the adult fish. The cycle lasts for 20 days.

[0044] S4, daily management: early and late inspection of the domestication pool, timely cleaning of the residual bait, water change every 4 days, with a water change amount of 70%.

[0045] The artificial bait includes the following raw materials in parts by weight: corn flour 30 parts, fish bone powder 25 parts, spirulina powder 17 parts, yao column powder 6 parts, grape seed powder 12 parts, red garlic 4 parts, taurine 5.5 parts, and additives 3 parts. The additives are composed of luteolin, limonin, gastrodin polysaccharide, and gingerol in a mass ratio of 8:5:18:0.6.

[0046] Comparative Example 1

[0047] The difference between this comparative example and Example 3 is that the bait domestication, temperature domestication, and sound wave domestication are all completed in one domestication pool.

[0048] Comparative Example 2

[0049] The difference between this comparative example and Example 3 is that the bait domestication in the juvenile stage is not performed in the domestication step, and the young fish is directly subjected to temperature domestication.

[0050] Comparative Example 3

[0051] The difference between this comparative example and Example 3 is that the temperature domestication of the young fish is not performed in the domestication step, and the young fish is continuously subjected to bait domestication and then sound wave domestication.

[0052] Comparative Example 4

[0053] The difference between this comparative example and Example 3 is that the temperature domestication is constant temperature domestication, with a water temperature of 33℃ and water flow stimulation for 60 minutes, and the continuous domestication lasts for 2 weeks.

[0054] Comparative Example 5

[0055] The difference between this comparative example and Example 3 is that the artificial bait includes the following raw materials in parts by weight: corn flour 50 parts, fish bone powder 33 parts, spirulina powder 22 parts, yao column powder 8 parts, grape seed powder 2 parts, red garlic 1 part, taurine 0.2 parts, and additives 1 part.

[0056] Comparative Example 6

[0057] The difference between this comparative example and Example 3 is that the artificial bait does not contain additives.

[0058] Test Example 1 - Anti-heat stress test

[0059] Ten groups of net cages were set up, corresponding to the above-mentioned Example 1-3 and Comparative Example 1-6, and a control group, with 3 replicates in each group. The above-mentioned domesticated golden pompanos were placed in the net cages for further breeding. The water temperature in the net cages was set to 35±2℃, the water quality pH value was 8, the dissolved oxygen was >5 mg / L, the ammonia nitrogen was <0.2 mg / L, the salinity was 30, the transparency was 80 cm, the nitrite content was 0.035 mg / L, and the feed was fed according to normal feeding management. After 48 h of stress, the survival rate, weight loss rate, and biochemical index of the golden pompanos were observed.

[0060] 1. Weight loss rate = (M-m) / M x 100%, wherein M is the sample fish weight before stress, and m is the sample fish weight after stress.

[0061] 2. Serum biochemical index determination: alanine aminotransferase (ALT), aspartate aminotransferase (AST) activity, and cortisol content were tested using an activity detection kit.

[0062] 3. Liver biochemical index determination: the liver sample was homogenized with phosphate buffered saline (pH 7.4) at a ratio of 1:9 in an ice bath, then centrifuged at 4℃, 3000 r / min for 15 min, and the supernatant was taken. The total superoxide dismutase (SOD) in the liver, total antioxidant capacity (T-AOC) in the liver, glutathione peroxidase (GSH-Px) in the liver, glutathione reductase (GR) in the liver, and malondialdehyde (MDA) in the liver were tested according to the activity detection kit.

[0063] The activity before and after stress was compared to calculate the activity growth rate.

[0064] Table 1:

[0065]

[0066] Table 2:

[0067]

[0068] The above results show that the ALT and AST activities in the serum of the golden pompanos in the comparative example group and the control group after stress were significantly higher than before stress, indicating that the liver cells of the fish were damaged by oxidative stress, the stability of the cell membrane was destroyed, ALT and AST were released into the blood, and the blood Cor content increased significantly. The example group has good anti-stress ability, and the Cor change rate is small after 48 h of stress, effectively reducing the stress regulation hormone cortisol content and the basal metabolic level, passivating the stress response ability of the golden pompano, and accelerating the growth of the fish body.

[0069] Table 3:

[0070]

[0071] The above results show that after the domestication and breeding of the golden pompano according to the application, the activities of total superoxide dismutase (SOD) in the liver, total antioxidant capacity (T-AOC) in the liver, glutathione peroxidase (GSH-Px) in the liver, and glutathione reductase (GR) in the liver are significantly increased, and the generation amount of malondialdehyde (MDA) is reduced, so that excessive reactive oxygen species (ROS) can be removed, the antioxidant stress resistance of the body is enhanced, and the oxidative damage of the body caused by heat stress is reduced.

[0072] The content of MDA in the liver is significantly higher than that before stress, indicating that heat stress induces oxidative stress damage of the liver of the golden pompano. The change of the content of MDA in the liver of the example group is smaller than that of the comparison example group, indicating that the example group has a certain stress resistance, the generation and removal of free radicals in the body are in a relatively balanced state, while the free radicals with strong oxidation in the comparison example group and the control group will damage the plasma membrane, cause lipid peroxidation, produce lipid peroxide (MDA), and MDA will increase the rigidity of the membrane and reduce the fluidity of the membrane, so that the structure and function of the membrane are damaged, indirectly reflecting the degree of damage of the cells in the body.

[0073] The above only describes the preferred embodiments of the application and is not intended to limit the application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A method for the culture of goldfish (Carassius auratus) against heat stress, characterized by: The gold pompano fry is put into the acclimation pool I, the acclimation pool II and the acclimation pool III, and the gold pompano is acclimated according to the juvenile stage, the young fish stage and the adult fish stage, and the specific steps include the following steps. S1, feeding domestication: select healthy fry into domestication pool I, fry density is 150-180 thousand tails / m 3 , feeding artificial bait, feeding 3-5 times a day, feeding amount is 2-5% of fry weight, stop feeding for 2 days after continuous feeding for 5 days, and feed in turn until the juvenile stage; S2, temperature acclimation: the above juvenile fish are put into the acclimation pool II, and the juvenile fish are put at a density of 220-280 tails / m 3 , and acclimated according to four temperature segments, the first segment is set to water temperature 22-25℃, water flow stimulation for 15-20min, the second segment is set to water temperature 26-30℃, water flow stimulation for 20-25min, the third segment is set to water temperature 31-35℃, water flow stimulation for 11-14min, the fourth segment is set to water temperature 36-40℃, water flow stimulation for 5-10min, and continuously acclimated for 2 weeks; S3, sound wave domestication: after temperature domestication, the young fish is grown into adult fish, and is transported to the domestication pool III with a density of 100-120 tails / m 3 The fish population is driven by sound waves every 8-14 hours per day, each driving lasts for 150-300 seconds, and the sound is emitted while the artificial bait is uniformly fed, and the cycle lasts for 18-22 days. S4, daily management: early and late patrol the acclimation pool I, the acclimation pool II and the acclimation pool III, clean up the residual thread in time, and replace water every 3-5 days, and the water replacement amount is 50-80%; The red light illumination management is simultaneously carried out in the acclimation of S1-S3, is started 0.5-1h before each time of feeding the artificial mixed feed, the illumination intensity is 600-1000lx, and the illumination time is 1-3h / d; The artificial mixed feed includes the following raw materials in parts by weight: corn flour 20-50 parts, fish bone powder 12-33 parts, spirulina powder 13-22 parts, yao column powder 5-8 parts, grape seed powder 8-15 parts, red skin garlic 2-6 parts, taurine 1.2-8.5 parts and additives 2-4 parts; The additives are composed of luteolin, limonin, gastrodin polysaccharide and gingerol in a mass ratio of (6-10):(3-8):(12-25):(0.3-0.9).

2. A method of rearing goldfish against heat stress as claimed in claim 1, wherein: The water quality management conditions in the acclimation pool I, the acclimation pool II and the acclimation pool III are as follows: pH value 7.6-8.2, dissolved oxygen >5mg / L, ammonia nitrogen <0.2mg / L, salinity 22-36, transparency 70-90cm, nitrite content 0.021-0.058mg / L.

3. A method of rearing goldfish against heat stress as claimed in claim 1, wherein: The S2 in every other day morning 8:00-9:00 and afternoon 3:00-4:00 temperature acclimation, control water flow rate is 0.8-1.5 m 3 / h.

4. A method of rearing goldfish against heat stress as claimed in claim 1, wherein: After each temperature acclimation in S2 is finished, the artificial mixed feed is fed, and the feeding amount is 5-10% of the weight of the young fish.

5. A method of rearing goldfish against heat stress as claimed in claim 1, wherein: The sound wave frequency in S3 is 900-1100Hz.

6. A method of rearing goldfish against heat stress as claimed in claim 1, wherein: The feeding amount in S3 is 6-18% of the weight of the adult fish.

7. A method of rearing goldfish against heat stress as claimed in claim 1, wherein: The artificial mixed feed includes the following raw materials in parts by weight: corn flour 33 parts, fish bone powder 25 parts, spirulina powder 18 parts, yao column powder 7 parts, grape seed powder 12 parts, red skin garlic 4 parts, taurine 6.2 parts and additives 3 parts.

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