A method and application of cultivating greater amberjack seedlings in indoor cement pond

Abstract

The present invention discloses a method for cultivating greater amberjack seedlings in an indoor cement pool, comprising the following steps: fertilized egg hatching, environmental regulation, bait and feeding methods, net-pulling training, and fish screening and pool division. The method adopts cement pool cultivation, and through water quality regulation, water flow environment regulation, and light environment regulation, selects nutritious oyster fertilized eggs instead of rotifers as the starter bait for fry, and through step-by-step net-pulling training, and cultivating seedlings of different specifications in different pools, the hatching rate of greater amberjack fertilized eggs and the survival rate of newly hatched fry are improved. The present invention also provides the use of the above method in improving the hatching rate of greater amberjack fertilized eggs, improving the weight, total length, survival rate, and yield rate of greater amberjack fry, improving the expression of genes related to muscle growth and development of greater amberjack, improving the microbial species coverage, richness, and diversity of greater amberjack intestinal flora, regulating the composition structure of greater amberjack intestinal microbial colonies, and increasing the proportion of beneficial bacteria in the flora or improving intestinal health.

Description

Technical Field

[0001] The invention belongs to the technical field of marine fish breeding, and particularly relates to a method and application of cultivating greater amberjack seedlings in an indoor cement pool. Background Art

[0002] The greater amberjack (Seriola dumerili) belongs to the genus Seriola of the family Seriola in the order Perciformes. It is a deep-sea economic fish that grows fast, is rich in nutrition, tastes delicious, and has a broad market. However, the artificial breeding technology of greater amberjack was not mature in the past, and there was no stable source of seedlings, which limited the development of the greater amberjack aquaculture industry. At present, there have been breakthroughs in the artificial breeding technology of greater amberjack, which has laid the foundation for the development of greater amberjack seedling cultivation technology.

[0003] The high-position pond culture model is a high-yield, high-profit, and easy-to-manage culture model. However, the traditional high-position pond seedling cultivation model has a large water body and the water quality environment is difficult to control. The greater amberjack fry is relatively fragile and very sensitive to changes in the water quality environment, which leads to a low survival rate of the newly hatched fry in the early stage. The water body of the indoor cement pool is small, the water quality environment is easy to control, and is not easily affected by the external environment. Therefore, those skilled in the art are in urgent need of a stable and efficient greater amberjack seedling cultivation method to improve the hatching rate of greater amberjack fertilized eggs and the survival rate of newly hatched fry, thereby improving the efficiency of greater amberjack artificial breeding and reducing the cost of artificial breeding. Summary of the invention

[0004] The invention aims to provide a method for cultivating greater amberjack seedlings in an indoor cement pool. The method adopts cement pool cultivation, selects fertilized oyster eggs instead of rotifers as the starter bait for fry through water quality control, water flow environment control and light environment control, and improves the hatching rate of greater amberjack fertilized eggs and the survival rate of newly hatched fry through step-by-step net training and cultivating seedlings of different specifications in separate pools.

[0005] The present invention also aims to provide the use of the above method in improving the hatching rate of fertilized eggs of greater amberjack, increasing the weight, total length, survival rate and yield rate of juvenile greater amberjack, increasing the expression of genes related to muscle growth and development of greater amberjack, increasing the microbial species coverage, flora richness and flora diversity of the intestinal flora of greater amberjack, regulating the composition structure of the intestinal microbial colony of greater amberjack, increasing the proportion of beneficial bacteria in the flora and improving intestinal health.

[0006] The first object of the present invention can be achieved by the following technical scheme: a method for cultivating greater amberjack seedlings in an indoor cement pond, comprising the following steps: hatching fertilized eggs, environmental regulation, bait and feeding methods, net training and fish screening and pond separation;

[0007] A) Fertilized egg incubation: After removing the dead eggs, the fertilized eggs are evenly distributed in an indoor cement pool with an area of ​​15-30m2 The spawning depth is 50-60cm, and the spawning density is 10,000-15,000 eggs / m 3 The density of air stone is 0.5-1 / m 2 , inflate to make the water surface slightly boil, use local seawater, and keep the water temperature at 23.0-23.5℃;

[0008] B) Environmental regulation: The environmental regulation includes water quality regulation, water flow environment regulation and light environment regulation;

[0009] The water quality control refers to mixing Bacillus subtilis, Bacillus licheniformis and Lactobacillus before laying eggs, spraying the whole pond to cultivate water quality, adding 10-15cm of water every day until 100-110cm 1-4 days after laying eggs; starting from the 5th day, changing 10-15cm of water every day, and adding Chlorella vulgaris to stabilize the water quality and serve as biological bait; starting from the 9th day, changing 30-40cm of water every day; starting from the 14th day, conducting flow-through culture and suctioning the bottom once a day, changing 50-60cm of water every day; starting from the 20th day, changing the water once in the morning and evening every day, and changing 50-60cm of water each time; during the seedling cultivation process, the water quality condition is tested every day;

[0010] The water flow environment regulation refers to controlling the air stone from the 1st to 2nd day after spawning to make the water surface slightly boiling; from the 3rd day, 80% of the fry have emerged from the membrane, and the air stone inflation amount is reduced to make the water surface slightly fluctuate; from the 9th day, the fry have the ability to swim, and the inflation amount is increased to make the water surface boiling, so as to provide a suitable water flow environment and sufficient oxygen for the growth and development of the fry;

[0011] The light environment regulation refers to the light-proof treatment for 1-2 days after egg laying, with the light intensity not exceeding 500 lux; starting from the 3rd day, the light time is not less than 16 hours per day, and the light intensity is 1000-20000 lux, of which the light intensity is greater than 10000 lux for not less than 8 hours;

[0012] C) Feed and feeding method: According to different development stages, the feed is oyster fertilized eggs-rotifers-artemia nauplii-copepods-compound feed;

[0013] On the 1st to 2nd day after egg laying, the fertilized eggs are not fully hatched and no feeding is required; on the 3rd day after egg laying, the fry have all emerged from the membrane, and the fertilized oyster eggs are used as the opening bait, which is sprinkled throughout the pond, and each pond is fed with fertilized eggs produced by 5-6 oysters each time, twice a day; on the 6th day after egg laying, stop feeding fertilized oyster eggs and start feeding rotifers at a density of 1000-3000 / L; on the 14th day after egg laying, start feeding Artemia nauplii at a density of 300-600 / L, and reduce the feeding of rotifers, and gradually switch to Artemia nauplii; on the 25th day after egg laying, stop feeding rotifers, start feeding copepods, reduce the feeding of Artemia nauplii, and gradually switch to copepods; on the 31st day after egg laying, stop feeding Artemia nauplii, reduce the feeding of copepods, and start acclimatization with compound feeds;

[0014] D) Pulling net training: On the 23rd day after egg laying, catch-up training is carried out every day, and on the 28th day after egg laying, pulling net training is carried out every day;

[0015] E) Screening and sorting fish into ponds: 31 days after laying eggs, the fry grow to 2-4cm. There are large individual differences and the fry may cannibalize each other. They need to be sorted into ponds for cultivation until the fry grow to 6-7cm and then transferred to sea cages for subsequent cultivation.

[0016] In the above-mentioned method for cultivating greater amberjack seedlings in indoor cement ponds:

[0017] More preferably, the cement pool area in step A) is 15-20m 2 .

[0018] Optionally, the water conditions during the incubation of the fertilized eggs in step A) are: water temperature 23-23.5°C, dissolved oxygen ≥6 mg / L, pH 8.2-8.3, salinity 6-27, ammonia nitrogen <0.02 mg / L, and nitrite <0.01 mg / L.

[0019] Optionally, during the water quality control in step B), the volume ratio of Bacillus subtilis, Bacillus licheniformis and Bacillus lactis is 1:1:1, and the concentrations of the three Bacillus are all 5000-8000 / L, more preferably, the concentrations of the three Bacillus are all 6000 / L.

[0020] More preferably, in step B), the water quality control refers to mixing Bacillus subtilis, Bacillus licheniformis and Lactobacillus before egg laying, spraying the whole pond, cultivating water quality, adding 10 cm of water every day until 100 cm 1-4 days after egg laying; starting from the 5th day, changing 10 cm of water every day, and adding Chlorella vulgaris to stabilize the water quality and serve as biological bait; starting from the 9th day, changing 30 cm of water every day; starting from the 14th day, conducting flow-through culture and suctioning the bottom once a day, changing 50 cm of water every day; starting from the 20th day, changing the water once in the morning and evening every day, and changing 50 cm of water each time; during the seedling cultivation process, the water quality is tested every day.

[0021] Optionally, during the water quality control in step B), the water conditions during seedling cultivation are: water temperature 23-28°C, dissolved oxygen ≥6mg / L, pH 8.2-8.6, salinity 26-29, ammonia nitrogen <0.02mg / L, nitrite <0.01mg / L.

[0022] Furthermore, in step B), when the water quality is regulated, when the ammonia nitrogen and nitrite levels in the water are too high during the seedling cultivation process, ecological preparations including photosynthetic bacteria and / or Bacillus are used for regulation.

[0023] Optionally, during the water flow environment regulation in step B), starting from the third day, 80% of the fry have emerged from the membrane, the inflation amount of the air stone is reduced to make the water surface slightly fluctuate, and the water inlet is wrapped with a long cylindrical cloth bag to allow seawater to flow into the pool along the cloth bag, and the water outlet is wrapped with a 100-mesh silk net.

[0024] Optionally, the oyster fertilized eggs in step C) are obtained by mixing reproductive cells produced by male and female oysters in a ratio of 1:5 in seawater for 2-3 hours.

[0025] More preferably, the oyster fertilized eggs in step C) are obtained by mixing reproductive cells produced by male and female oysters in a ratio of 1:5 in seawater for 2 hours.

[0026] Optionally, on the 23rd day after spawning in step D), chasing training is carried out every day, the training process is to first lower the water level to 40-50cm, so that the fish school is in a state of stress, then gently splash the water surface with water to chase the fry, and continue for 20-40 minutes. After the end, the water level is restored to the original water level. 28 days after spawning, the water level is lowered to 20-30cm during training, and the fry are chased back and forth using a purse seine every day to encircle the fish school in a smaller area so that the fish school is in a high-density environment. The intensity is gradually increased according to the reaction of the fish school, and 20-40 minutes of net pulling training is carried out every day. The occurrence of partial convulsion state in the fish school is used as a signal to stop the training. After the end, the water level is restored to the original water level, so that the fish school gradually adapts to the high-density stress state, and prepares for the subsequent screening of fish and ponding.

[0027] More preferably, on the 23rd day after spawning in step D), chasing training is carried out every day, the training process is to first lower the water level to 50cm, so that the fish school is in a state of stress, then gently splash the water surface with water to chase the fry, and continue for 30 minutes. After the end, the water level is restored to the original water level. 28 days after spawning, the water level is lowered to 30cm during training, and the fry are chased back and forth using a purse seine every day to encircle the fish school in a smaller area so that the fish school is in a high-density environment. The intensity is gradually increased according to the reaction of the fish school, and the net pulling training is carried out for 30 minutes every day. The partial convulsion state of the fish school is used as a signal to stop the training, and the water level is restored to the original water level after the end, so that the fish school gradually adapts to the high-density stress state, and prepares for the subsequent screening of fish ponds.

[0028] Optionally, 31 days after laying eggs in step E), the fry grow to 2-4 cm, the individual differences are large, and the fry have cannibalism, so they need to be cultured in separate ponds. The fry obtained by pulling the net is transferred to a fish sieve, and the fish sieve floats on the water surface of a new culture pond. Smaller individuals automatically swim into the culture pond, and larger individuals are transferred to a larger mesh fish sieve or a new culture pond. In this way, a more suitable seedling raising strategy is adopted for fry of different sizes to promote the growth of fry, until the fry grow to 6-7 cm and are transferred to sea cages for subsequent cultivation.

[0029] Optionally, the fish sieve in step E) is a hollow hemispherical shape, the hemispherical surface is a mesh, and is made of stainless steel, and the edge of the hemispherical surface is a foam material, so that the fish sieve can float on the water surface.

[0030] The above-mentioned second object of the present invention can be achieved by the following technical scheme: the application of the above-mentioned method in improving the hatching rate of fertilized eggs of greater amberjack, increasing the weight, total length, survival rate and yield rate of greater amberjack fry, increasing the expression of genes related to muscle growth and development of greater amberjack, increasing the microbial species coverage, richness and diversity of the intestinal flora of greater amberjack, regulating the composition structure of the intestinal microbial colony of greater amberjack, increasing the proportion of beneficial bacteria in the flora or improving intestinal health.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) The present invention provides a method for cultivating greater amberjack seedlings in an indoor cement pool. According to the characteristics that greater amberjack fry are sensitive to changes in the water environment and are prone to large-scale mortality, the method does not use a hatching bucket during hatching, but directly places the fertilized eggs in a cement pool to prevent the seedlings from dying during the transfer process.

[0033] (2) In the indoor cement pond seedling cultivation method of the present invention, a gentle water flow environment and stable water conditions are provided for fry and juvenile fish. In particular, three beneficial bacteria, namely, Bacillus subtilis, Bacillus licheniformis and Lactobacillus, are mixed in a ratio of 1:1:1 to cultivate the water body, which can not only effectively decompose harmful substances such as organic waste, ammonia nitrogen, sulfide, etc., inhibit pathogenic microorganisms in the water body, maintain the ecological balance of the water body, but also promote the growth of fry and the development of immune organs, enhance the immunity of fry, and greatly reduce the mortality rate in the early stage of seedling cultivation;

[0034] (3) In the method for cultivating greater amberjack in an indoor cement pond, under natural conditions, extreme weather often leads to insufficient light, which affects the feeding activity of the fry. The present invention ensures the normal feeding behavior of the fry and promotes the growth and development of the fry by artificially controlling the intensity and duration of light.

[0035] (4) In the method for cultivating greater amberjack in an indoor cement pond, fertilized oyster eggs, which are more nutritious, are selected as the starter feed for the fry instead of rotifers, which is more conducive to the growth and development of the fry in the early stage. In addition, compared with the situation that rotifers are often out of supply during the peak period of fry cultivation, the source of fertilized oyster eggs is more stable.

[0036] (5) In the indoor cement pond seedling cultivation method of greater amberjack of the present invention, the stress resistance of the fry is enhanced through step-by-step net-pulling training;

[0037] (6) In the method for cultivating greater amberjack seedlings in an indoor cement pond of the present invention, seedlings of different specifications are cultivated in separate ponds, which reduces the occurrence of cannibalism and improves the survival rate of the seedlings;

[0038] (7) Compared with the method of cultivating greater amberjack seedlings in outdoor high-position ponds, the method of the present invention can improve the hatching rate of fertilized eggs of greater amberjack, increase the weight, total length, survival rate and hatching rate of greater amberjack fry, increase the expression of genes related to muscle growth and development of greater amberjack, increase the microbial species coverage, richness and diversity of the intestinal flora of greater amberjack, regulate the composition structure of the intestinal microbial colony of greater amberjack, increase the proportion of beneficial bacteria in the flora or improve the intestinal health of greater amberjack. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the muscle growth-related gene expression in Example 1 and Comparative Example 1;

[0040] Note: ns, no significant difference; *, significant difference (P < 0.05); N, indoor cement pool; W, outdoor high-level pool;

[0041] Figure 2 It is the PCoA analysis at the genus level based on the bray distance algorithm in Example 1 and Comparative Example 1;

[0042] Figure 3 is the UPGMA clustering tree of each group of samples in Example 1 and Comparative Example 1 at the genus level based on the Bray distance algorithm;

[0043] Figure 4 It is the intestinal flora composition of each group in Example 1 and Comparative Example 1 at the phylum and class levels. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific implementation modes. The following examples are only illustrative and not restrictive, and the protection scope of the present invention cannot be limited thereto.

[0045] Example 1

[0046] The method for cultivating greater amberjack seedlings in an indoor cement pond provided by the present invention comprises the following steps:

[0047] 1. Hatching of fertilized eggs:

[0048] Before laying eggs, potassium permanganate is used to spray and disinfect the entire cement pool;

[0049] After removing the turbid and whitish dead eggs, the fertilized eggs are evenly distributed in an indoor cement pool with an area of ​​15m 2 The egg-laying water depth is 60cm, and the density is 10,000-15,000 eggs / m 3 , 8 air stones are evenly arranged in each pool, and the water is slightly boiled by aeration, and the water used is local seawater;

[0050] The water conditions during the incubation period were: water temperature 23-23.5°C, dissolved oxygen ≥6 mg / L, pH 8.2, salinity 27, ammonia nitrogen <0.02 mg / L, and nitrite <0.01 mg / L.

[0051] 2. Environmental control:

[0052] Environmental regulation for seedling cultivation includes regulation of water quality, water flow environment, and light environment.

[0053] The water quality control process is as follows:

[0054] Before laying eggs, water quality should be cultivated. Bacillus subtilis, Bacillus licheniformis and Lactobacillus should be mixed and sprayed throughout the pond. The mixing ratio is 1:1:1 (volume ratio). The concentration of the three strains of Bacillus is 6000 / L.

[0055] 1-4 days after laying eggs, add 10cm of water every day until it reaches 100cm; starting from the 5th day, change 10cm of water every day, and add Chlorella to stabilize the water quality and serve as biological bait; starting from the 9th day, change 30cm of water every day; starting from the 14th day, carry out flow water culture and clean the bottom once a day, and change 50cm of water every day; starting from the 20th day, change the water once in the morning and evening every day, and change 50cm of water each time.

[0056] During the seedling cultivation process, the water conditions are: water temperature 23-28℃, dissolved oxygen ≥6mg / L, pH8.2-8.6, salinity 26-29, ammonia nitrogen <0.02mg / L, nitrite <0.01mg / L; when the ammonia nitrogen and nitrite levels in the water are too high, ecological agents such as photosynthetic bacteria and Bacillus are used for regulation.

[0057] The water flow environment regulation process is:

[0058] 1-2 days after laying the eggs, control the air stone to make the water surface slightly boiling. Starting from the 3rd day, 80% of the fry have hatched, reduce the amount of air stone inflation, make the water surface slightly fluctuate, and wrap the water inlet with a long cylindrical cloth bag to allow seawater to flow into the pool along the bag. Use a 100-mesh silk net to wrap the water outlet. The smaller mesh can reduce the water flow rate at the outlet to prevent the newly hatched fry from being sucked into the outlet and injured during the water change process. Starting from the 9th day, the fry have a certain swimming ability, increase the amount of inflation, make the water surface boiling, provide a water flow environment and sufficient oxygen for the growth and development of the fry.

[0059] The lighting environment control process is:

[0060] 1-2 days after laying eggs, protect the fish from light and keep the light intensity no more than 500 lux. Starting from the 3rd day, add a light source to provide supplementary light at night. Prolonging the light time will promote the feeding and growth of the fry. The light time should be no less than 16 hours a day, with a light intensity of 1000-20000 lux, of which the light intensity is greater than 10000 lux for no less than 8 hours.

[0061] 3. Bait and feeding method:

[0062] According to different development stages, the feeding sequence is oyster fertilized eggs - rotifers - Artemia nauplii - copepods - compound feed, including:

[0063] On the first or second day after laying, the fertilized eggs have not fully hatched and no feeding is needed. On the third day after laying, all the fry have emerged from the membranes and fertilized oyster eggs are used as the starter bait. The fertilized oyster eggs used are made from reproductive cells produced by male and female oysters in a ratio of 1:5, which are mixed in a small amount of seawater for 2 hours. Spray the whole pond, feed each pond with fertilized eggs produced by 5-6 oysters each time, twice a day; on the 6th day after egg laying, stop feeding fertilized oyster eggs and start feeding rotifers at a density of 1000-3000 / L; on the 14th day after egg laying, start feeding Artemia nauplii at a density of 300-600 / L, and reduce the feeding of rotifers, and gradually switch to Artemia nauplii; on the 25th day after egg laying, stop feeding rotifers, start feeding copepods, reduce the feeding of Artemia nauplii, and gradually switch to copepods; on the 31st day after egg laying, stop feeding Artemia nauplii, reduce the feeding of copepods, and start acclimatization with compound feed.

[0064] 4. Pulling net training:

[0065] On the 23rd day after spawning, daily chasing training was conducted. The training process was to first lower the water level to 50cm to put the fish in a state of stress, then go into the pond, gently splash the water surface with water, chase the fry, and continue for 30 minutes. After the training, the water level was restored to the original level. On the 28th day after spawning, the water level was lowered to 30cm during training. Every day, the fry were chased back and forth using a seine net in the pond, and the fish were enclosed in a smaller area, so that the fish were in a high-density environment. The intensity was gradually increased according to the reaction of the fish, and the net pulling training was conducted for 30 minutes every day. The partial convulsion state of the fish was used as a signal to stop the training, and the water level was restored to the original level after the training. In this way, the fish gradually adapted to the high-density stress state and prepared for the subsequent screening and sorting of fish.

[0066] 5. Screening fish and dividing the ponds:

[0067] 31 days after laying eggs, the fry grow to 2-4cm, with large individual differences. There is cannibalism among the fry, so they need to be cultured in separate ponds. The fry obtained from the pulling net are transferred to the fish sieve using a hand net. The fish sieve used is a hollow hemispherical screen with mesh on the hemispherical surface. It is made of stainless steel and the edge of the hemispherical surface is made of foam. The fish sieve floats on the water surface of the new breeding pond. Smaller individuals automatically swim into the breeding pond, while larger individuals are transferred to larger mesh fish sieves or new breeding ponds. In this way, more suitable breeding strategies are adopted for fry of different sizes to promote the growth of fry until they grow to 6-7cm and are transferred to sea cages for subsequent cultivation.

[0068] Example 2

[0069] The difference from Example 1 is that the cement pool area is 20m 2 .

[0070] Example 3

[0071] The difference from Example 1 is that in step B), when the water quality is controlled, the volume ratio of Bacillus subtilis, Bacillus licheniformis and Lactobacillus is 1:1:1, and the concentration of the three Bacillus is 6500 / L.

[0072] Comparative Example 1

[0073] Different from Example 1, in this Comparative Example 1, the method for cultivating greater amberjack seedlings in a high-position pond was adopted to cultivate greater amberjack seedlings.

[0074] In this comparative example 1, a hatching bucket was used to hatch the fertilized eggs of greater amberjack, and the fertilized eggs were moved to a high-level pond for cultivation after hatching; the water body was not cultivated with three beneficial bacteria, namely Bacillus subtilis, Bacillus licheniformis and Lactobacillus; the starting bait was changed from oyster fertilized eggs to rotifers; the lighting environment was natural light; there was no periodic net pulling training, and other seedling cultivation conditions were the same as those in the seedling cultivation method of the greater amberjack high-level pond.

[0075] The methods in Example 1 and Comparative Example 1 were used in the cultivation of greater amberjack seedlings:

[0076] 1. Test methods

[0077] 1.1. Breeding of greater amberjack seedlings

[0078] On May 31, 2022, at the Hongda Aquaculture Base in Zhangpu County, Zhangzhou, Fujian, the greater amberjack fry cultivation method in the above-mentioned Example 1 and Comparative Example 1 was used to cultivate greater amberjack fry for 31 days. In Example 1 and Comparative Example 1, 500,000 fertilized greater amberjack eggs were released respectively.

[0079] In Example 1, 500,000 fertilized eggs were placed in 4 indoor cement pools and incubated and cultured according to the above method.

[0080] Meanwhile, in Comparative Example 1, 500,000 fertilized eggs were placed in an incubation bucket at a density of 150,000 to 180,000 eggs / m 3 After hatching, the fish are transferred to outdoor high-level pools. There are 3 pools in total, with an area of ​​130-170m 2 , the stocking density is 1500-2000 / m 3 , using traditional high-level pond seedling cultivation methods for cultivation.

[0081] 1.2. Measurement of growth parameters and sample collection

[0082] On June 3, the number of fertilized eggs of greater amberjack that had hatched was counted.

[0083] On July 1, most of the fry in the indoor cement pool (N) and outdoor high-level pool (W) had grown to 2-4cm, with large individual differences and cannibalism among the fry, so they needed to be cultivated in separate pools.

[0084] Before the ponds were divided, 10 young greater amberjacks were randomly picked from each pond in the indoor cement pond and the outdoor high-level pond, and their growth performance-related parameters were statistically analyzed and sampled. Muscle tissue was placed in a 2mL enzyme-free centrifuge tube at -80°C to measure the expression of growth-related genes. Whole fish samples were placed in a 2mL enzyme-free centrifuge tube at -80°C to measure the composition of their intestinal flora. Growth performance indicators such as hatching rate, total length, weight, survival rate, and yield rate were calculated based on statistical data, including:

[0085] Hatching rate = total number of hatched fry / total number of fertilized eggs × 100%;

[0086] Survival rate = total number of surviving fry / total number of hatched fry × 100%;

[0087] Survival rate = total number of fry with a total length of 2.8-3.3 cm / total number of surviving fry × 100%.

[0088] 1.3. Total RNA extraction and cDNA synthesis

[0089] Muscle tissue of juvenile greater amberjack was cut and total RNA from muscle was extracted using the Trizol method. Agarose gel electrophoresis was used to detect RNA sample degradation and DNA contamination. The purity and concentration of RNA were detected by Nanodrop 2000 spectrophotometer. The total RNA from muscle tissue was reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Japan), and the cDNA was stored in a -20°C refrigerator for subsequent fluorescence quantitative PCR (qPCR).

[0090] 1.4. Primer synthesis and qPCR detection of muscle growth-related genes

[0091] Primers synthesized by Shanghai Bioengineering Co., Ltd. (Shanghai, China) were designed using Primer5.0 (Table 1). qPCR was performed using the SYBR Green qPCR Mix (with ROX) kit (Guangzhou Dongsheng Biotechnology Co., Ltd.) and the experimental instrument was a Light Cycler 480 fluorescence quantitative PCR instrument. β-actin was used as the internal reference gene, and the relative expression of the gene was analyzed using the 2-ΔΔCt method. T-test significance analysis was performed using Graphpad Prism 9 software, and the data were expressed as mean ± standard deviation. When P < 0.05, the difference was significant; when P < 0.01, the difference was extremely significant.

[0092] Table 1 Primers for genes related to muscle growth

[0093]

[0094]

[0095] 5. 16S sequencing of intestinal flora

[0096] After extracting genomic DNA from the sample, the V3+V4 region of 16S rDNA was amplified using specific primers with barcodes.

[0097] The primer sequences are:

[0098] 341F:CCTACGGGNGGCWGCAG;

[0099] 806R:GGACTACHVGGGTATCTAAT.

[0100] The purified amplification product (i.e., amplicon) was connected to a sequencing adapter to construct a sequencing library, and then sequenced on an Illumina machine. The above operations were completed by Guangzhou Kidio Biotechnology Co., Ltd.

[0101] 2. Experimental results

[0102] 2.1 Growth performance comparison

[0103] The effects of different cultivation modes of Example 1 and Comparative Example 1 on the growth performance of greater amberjack are shown in Table 2 below.

[0104] Table 2 Effects of different culture modes on the growth performance of greater amberjack

[0105] Group Hatching rate / % Weight / g Total length / cm Survival rate / % Success rate / % Example 1 Indoor cement pool 91.93±1.42 <![CDATA[0.51±0.04 * ]]> <![CDATA[3.27±0.09 * ]]> <![CDATA[40.97±4.63 * ]]> <![CDATA[75.47±2.26 * ]]> Comparative Example 1 Outdoor high-level pool 86.43±2.54 0.32±0.01 2.93±0.03 12.17±2.00 55.20±2.51

[0106] Note: *, represents significant difference (P<0.05)

[0107] The results in Table 2 show that the hatching rate of fertilized eggs and the weight, total length, survival rate and survival rate of greater amberjack fry obtained by the indoor cement pond fry cultivation method are higher than those of the outdoor high-position pond fry cultivation method, among which their weight, total length, survival rate and survival rate are significantly higher than those of the outdoor high-position pond fry cultivation method.

[0108] The results show that compared with the greater amberjack outdoor high-position pond seedling cultivation method, the greater amberjack indoor cement pond seedling cultivation method of the present invention can obtain a higher greater amberjack fertilized egg hatching rate and improve the greater amberjack fry weight, total length, survival rate and success rate.

[0109] 2.2 Expression of growth-related genes

[0110] Depend on Figure 1 It can be seen that the GH-IGFs growth axis and MRFs (myogenic regulatory factors) are involved in the regulation of muscle growth and development. Among them, high expression of ghr and igf2 can promote fish muscle growth and functional maturation; high expression of myf5 and myf6 can promote myoblast proliferation and myogenic differentiation; Figure 1 It can be seen that the expression of muscle growth-related genes such as myf5, myf6, igf2, and ghr in the muscle was significantly higher in the indoor cement pool than in the outdoor high-level pool (P < 0.05).

[0111] The results show that the method for cultivating greater amberjack seedlings in an indoor cement pond of the present invention can improve the expression of genes related to muscle growth and development of greater amberjack.

[0112] 2.3 Composition and structure of intestinal flora

[0113] 2.3.1 Microbial diversity

[0114] The effects of different cultivation modes on the diversity of intestinal flora of greater amberjack are shown in Table 3.

[0115] Table 3 Effects of different cultivation modes on the diversity of intestinal flora of greater amberjack

[0116]

[0117] Note: N: indoor cement pool; W: outdoor high-level pool.

[0118] As shown in Table 3, the coverage of each group of samples is higher than 99%, indicating that the experimental data has covered most microbial species. The Chao1 index is often used to measure sample abundance. The higher the Chao1 index, the higher the sample abundance. The Shannon and Simpson indices are used to measure bacterial diversity. The higher the index, the higher the diversity. According to the T-test, the Shannon index and Simpson index of the indoor cement pool group were significantly higher than those of the outdoor high-level pool group (P < 0.05), and there was no significant difference in the Chao1 index between the two groups (P > 0.05). This shows that the bacterial diversity of the indoor cement pool group is significantly higher than that of the outdoor high-level pool, and there is no significant difference in the richness of the bacterial community between the two groups.

[0119] The results showed that the indoor cement pond seed cultivation method for greater amberjack can not only make the cultivated greater amberjack have high microbial species coverage and microbial richness, but also improve the microbial diversity of greater amberjack.

[0120] 2.3.2 Comparative analysis of multiple samples

[0121] Principal coordinates analysis (PCoA) can be used to compare the similarities and differences in colony structures. The more similar the species composition of the samples, the closer the distance in the PCoA graph. Figure 2 It can be seen that the distance between the two groups of samples is relatively far, indicating that the similarity of the composition structure of the two groups of microbial colonies is low. UPGMA cluster analysis (based on Bray distance algorithm) was performed on each group of samples to construct a sample clustering tree ( Figure 3), the results showed that group W and group N were clustered into one branch, which was consistent with the results of PCoA analysis.

[0122] The results showed that the composition structure of microbial colonies was different between the two groups, indicating that the indoor cement pond seedling cultivation method of greater amberjack can adjust the proportion of microbial colony composition structure.

[0123] 2.3.3 Composition and structure of bacterial flora

[0124] Depend on Figure 4 As shown in Figure A, the taxonomic analysis of the samples in each group at the phylum level showed that the dominant phyla of the flora in groups N and W were Proteobacteria, Firmicutes, and Bacteroidetes. These three phyla are common phyla of intestinal flora and play an important role in maintaining the ecological balance of the intestinal flora of organisms. Proteobacteria is a common class of intestinal flora and helps maintain the intestinal environment; Bacillus (Firmicutes) has strong resistance to external harmful factors, can inhibit pathogens and promote biological growth; Bacteroidetes can participate in metabolic activities such as intestinal carbohydrate fermentation, utilization of nitrogen-containing substances, and biotransformation of bile acids and other steroids, and has an effect on the growth and development and immune regulation of organisms.

[0125] Depend on Figure 4 As shown in Figure B, the dominant classes of bacteria in group N are Gammaproteobacteria, Bacilli, and Bacteroidia, and the abundance of these three classes in group N is significantly higher than that in group W. The high expression of Proteobacteria, Bacilli, and Bacteroidia indicates the intestinal health of group N.

[0126] The results showed that the indoor cement pond seed cultivation method for greater amberjack can regulate the intestinal flora composition of greater amberjack, increase the proportion of beneficial bacteria in the flora, and improve intestinal health.

[0127] The present invention may be summarized in other specific forms that do not violate the spirit or main features of the present invention. The above embodiments of the present invention can only be regarded as an explanation of the present invention rather than a limitation. Therefore, any minor modifications, equivalent changes and modifications made to the above embodiments based on the essential technology of the present invention are within the scope of the technical solution of the present invention.

Claims

1. A method for cultivating greater amberjack seedlings in an indoor cement pond, characterized in that The following steps are involved: Fertilized egg hatching, environmental control, bait and feeding methods, net training and fish screening and pond sorting; A) Fertilized egg incubation: After removing the dead eggs, the fertilized eggs are evenly distributed in an indoor cement pool with an area of ​​15-30m 2 The spawning depth is 50-60cm, and the spawning density is 10,000-15,000 eggs / m 3 The density of air stone is 0.5-1 / m 2 , inflate to make the water surface slightly boil, use local seawater, and keep the water temperature at 23.0-23.5℃; B) Environmental regulation: The environmental regulation includes water quality regulation, water flow environment regulation and light environment regulation; The water quality control refers to mixing Bacillus subtilis, Bacillus licheniformis and Lactobacillus before laying eggs, spraying the whole pond to cultivate water quality, adding 10-15cm of water every day until 100-110cm 1-4 days after laying eggs; starting from the 5th day, changing 10-15cm of water every day, and adding Chlorella vulgaris to stabilize the water quality and serve as biological bait; starting from the 9th day, changing 30-40cm of water every day; starting from the 14th day, conducting flow-through culture and suctioning the bottom once a day, changing 50-60cm of water every day; starting from the 20th day, changing the water once in the morning and evening every day, and changing 50-60cm of water each time; during the seedling cultivation process, the water quality condition is tested every day; The water flow environment regulation refers to controlling the air stone from the 1st to 2nd day after spawning to make the water surface slightly boiling. From the 3rd day, 80% of the fry have emerged from the membrane, and the air stone inflation volume is reduced to make the water surface slightly fluctuate. From the 9th day, the fry have the ability to swim, and the inflation volume is increased to make the water surface boiling, so as to provide a suitable water flow environment and sufficient oxygen for the growth and development of the fry. The light environment regulation refers to the light-proof treatment for 1-2 days after egg laying, with the light intensity not exceeding 500 lux; starting from the 3rd day, the light time is not less than 16 hours per day, and the light intensity is 1000-20000 lux, of which the light intensity is greater than 10000 lux for not less than 8 hours; C) Feed and feeding method: According to different development stages, the feed is oyster fertilized eggs-rotifers-artemia nauplii-copepods-compound feed; On the 1st to 2nd day after egg laying, the fertilized eggs are not fully hatched and no feeding is required; on the 3rd day after egg laying, the fry have all emerged from the membrane, and the fertilized oyster eggs are used as the opening bait, which is sprinkled throughout the pond, and each pond is fed with fertilized eggs produced by 5-6 oysters each time, twice a day; on the 6th day after egg laying, stop feeding fertilized oyster eggs and start feeding rotifers at a density of 1000-3000 / L; on the 14th day after egg laying, start feeding Artemia nauplii at a density of 300-600 / L, and reduce the feeding of rotifers, and gradually switch to Artemia nauplii; on the 25th day after egg laying, stop feeding rotifers, start feeding copepods, reduce the feeding of Artemia nauplii, and gradually switch to copepods; on the 31st day after egg laying, stop feeding Artemia nauplii, reduce the feeding of copepods, and start acclimatization with compound feeds; D) Pulling net training: On the 23rd day after egg laying, chase training is carried out every day, and on the 28th day after egg laying, pull net training is carried out every day; E) Screening and sorting fish: 31 days after spawning, the fry grow to 2-4cm. There are large individual differences, and the fry may cannibalize each other. They need to be sorted and cultured in different ponds until the fry grow to 6-7cm, and then transferred to sea cages for subsequent culture. In step A), the water conditions during the incubation of fertilized eggs are: water temperature 23-23.5°C, dissolved oxygen ≥6 mg / L, pH 8.2-8.3, salinity 6-27, ammonia nitrogen <0.02 mg / L, nitrite <0.01 mg / L; During the water quality control in step B), the volume ratio of Bacillus subtilis, Bacillus licheniformis and Bacillus lactis is 1:1:1, and the concentration of the three Bacillus is 5000-8000 / L; The oyster fertilized eggs in step C) are obtained by mixing reproductive cells produced by male and female oysters in a ratio of 1:5 in seawater for 2-3 hours.

2. The method for cultivating greater amberjack seedlings in indoor cement ponds according to claim 1, characterized in that: In step A), the water conditions during the incubation of the fertilized eggs are: water temperature 23-23.5°C, dissolved oxygen ≥6 mg / L, pH 8.2-8.3, salinity 6-27, ammonia nitrogen <0.02 mg / L, and nitrite <0.01 mg / L.

3. The method for cultivating greater amberjack seedlings in indoor cement ponds according to claim 1, characterized in that: In step B), when the water quality is controlled, the water conditions during the seedling cultivation process are: water temperature 23-28°C, dissolved oxygen ≥6mg / L, pH 8.2-8.6, salinity 26-29, ammonia nitrogen <0.02mg / L, nitrite <0.01mg / L.

4. The method for cultivating greater amberjack seedlings in indoor cement ponds according to claim 1, characterized in that: In step B), when the water quality is regulated, when the ammonia nitrogen and nitrite levels in the water are too high during the seedling cultivation process, ecological preparations including photosynthetic bacteria and / or Bacillus are used for regulation.

5. The method for cultivating greater amberjack seedlings in indoor cement ponds according to claim 1, characterized in that: During the water flow environment regulation in step B), starting from the third day, when 80% of the fry have emerged from the membrane, the air stone inflation volume is reduced to make the water surface slightly fluctuate, and the water inlet is wrapped with a long cylindrical cloth bag to allow seawater to flow into the pool along the cloth bag, and the water outlet is wrapped with a 100-mesh silk net.

6. The method for cultivating greater amberjack seedlings in indoor cement ponds according to claim 1, characterized in that: On the 23rd day after spawning in step D), chasing training is carried out every day. The training process is to first lower the water level to 40-50 cm to put the fish in a state of stress, then gently splash the water surface with water to chase the fry for 20-40 minutes, and then return to the original water level after the end. 28 days after spawning, the water level is lowered to 20-30 cm during training, and the fry are chased back and forth with a purse seine every day to encircle the fish in a smaller area so that the fish are in a high-density environment. The intensity is gradually increased according to the reaction of the fish, and the net pulling training is carried out for 20-40 minutes every day. The occurrence of partial convulsions in the fish is used as a signal to stop the training. After the end, the water level is restored to the original water level, so that the fish gradually adapt to the high-density stress state and prepare for the subsequent screening and ponding.

7. The method for cultivating greater amberjack seedlings in indoor cement ponds according to claim 1, characterized in that: 31 days after the eggs are laid in step E), the fry grow to 2-4 cm, the individual differences are large, and the fry cannibalize each other, so they need to be cultured in separate ponds. The fry obtained by pulling the net are transferred to a fish screen, which floats on the water surface of a new culture pond. Smaller individuals automatically swim into the culture pond, and larger individuals are transferred to a fish screen with a larger mesh or a new culture pond. In this way, a more suitable seedling breeding strategy is adopted for fry of different sizes to promote the growth of fry, until the fry grow to 6-7 cm and are transferred to sea cages for subsequent cultivation; the fish screen is a hollow hemispherical shape, the hemispherical surface is a mesh, and is made of stainless steel. The edge of the hemispherical surface is a foam material, so that the fish screen can float on the water surface.

8. Use of the method according to any one of claims 1 to 7 in improving the hatching rate of fertilized eggs of greater amberjack, increasing the weight, total length, survival rate and hatching rate of greater amberjack fry, increasing the expression of genes related to muscle growth and development of greater amberjack, increasing the microbial species coverage, richness and diversity of the intestinal flora of greater amberjack, regulating the composition structure of the intestinal microbial colonies of greater amberjack, increasing the proportion of beneficial bacteria in the flora or improving intestinal health.

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