阅读说明：本技术 一种斑石鲷陆海接力养殖方法 (Luhai relay breeding method for oplegnathus punctatus ) 是由 贾玉东 高云涛 谢婷 张秉智 赵侠 关长涛 于 2021-07-14 设计创作，主要内容包括：本发明提供一种斑石鲷陆海接力养殖方法,是在每年5月底6月初将陆基工厂化循环水中养殖的斑石鲷通过活鱼运输船转运到网箱和/或围栏进行养殖；且在进行转运前投喂富含甘氨酸、赖氨酸和蛋氨酸的饲料。本发明通过在转运前对斑石鲷投喂富含甘氨酸、赖氨酸和蛋氨酸的饲料的,从而有效降低转运对斑石鲷的胁迫应激,缩短工厂化养殖斑石鲷转运后对自然生态环境的适应时间,促进其正常摄食,从而能够更快的恢复生长,同时在饲料中添加能表达抗菌肽的酵母菌,可提高其非特异性免疫能力,增强对环境抗逆能力。(The invention provides a method for relay culture of oplegnathus punctatus in the land and sea, which is characterized in that oplegnathus punctatus cultured in land-based industrial circulating water is transported to a net cage and/or a fence for culture by a live fish transport ship at the beginning of 6 months at the end of 5 months every year; and feeding the feed rich in glycine, lysine and methionine before the transfer. According to the invention, the feed rich in glycine, lysine and methionine is fed to the oplegnathus maculatus before transportation, so that the stress of transportation to the oplegnathus maculatus is effectively reduced, the adaptation time of the factory-cultured oplegnathus maculatus to the natural ecological environment after transportation is shortened, and the normal ingestion of the oplegnathus maculatus is promoted, so that the growth can be recovered more quickly, and meanwhile, the yeast capable of expressing the antibacterial peptide is added into the feed, so that the non-specific immunity of the oplegnathus maculatus can be improved, and the stress resistance to the environment is enhanced.)

1. A method for relay culture of oplegnathus punctatus on land and sea is characterized in that the oplegnathus punctatus cultured on land is transported to a net cage and/or a fence for culture at the beginning of 6 months at the bottom of 5 months; and feeding the feed rich in glycine, lysine and methionine before the transfer.

2. The method of claim 1 wherein the feed is fed glycine, lysine and methionine rich feed 10-15 days prior to transport.

3. The method of claim 1, wherein the feed is supplemented with a yeast capable of expressing the antimicrobial peptide.

4. The method of claim 4, wherein the amino acid sequence of said antimicrobial peptide is SEQ ID NO 1.

5. The method of claim 1, wherein the feeding is stopped not less than 16 hours prior to the transport of the oplegnathus fasciatus.

6. The method of claim 1, wherein the weight of the transported oplegnathus punctatus is not less than 90 g.

7. The method of claim 1, wherein the water temperature is maintained at 19-25 ℃ and the dissolved oxygen in the water is maintained at 7mg/L or more during transportation.

Technical Field

The invention belongs to the technical field of aquaculture, and particularly relates to a rock bream terrestrial sea relay culture method.

Background

Oplegnathus punctatus (Oplegnathus punctatus) belongs to the tropical offshore fish, is widely distributed in the yellow sea, east sea and south sea of China, and peripheral sea areas such as the Korean peninsula, the Japanese peninsula and the Chinese Taiwan island, and has the advantages of fine and smooth meat quality, rich nutrition, high growth speed and the like. Meanwhile, the market price is stable at 160-200 yuan/kg, the economic benefit is remarkable, and the feed is popular with breeding manufacturers in Shandong, Jiangsu, Fujian and Guangdong coastal areas at present, and the industrialization prospect is wide.

Land-based industrial circulating water and deep open sea large-scale net cages/fences are two main modes for culturing the oplegnathus fasciatus at present. The land-based factory circulating water culture mode has the advantages of water conservation, land conservation, accurate regulation and control of environmental factors such as water temperature, dissolved oxygen and the like, can not be influenced by external environment, realizes annual culture, and maintains higher operation energy consumption cost of the whole culture system. And the large-scale net cages and fences in deep and open sea trap culture water is large in volume, the moving space of culture objects is large, the culture water is sufficient, the water exchange is sufficient, the utilization rate of residual baits is high, the natural endowment of sea areas is fully utilized, the growth speed is high, meanwhile, the quality is closer to natural ecology, but the dissolved oxygen of the culture water environment temperature is not artificially controlled, and the whole-year culture cannot be realized. The oplegnathus fasciatus belongs to warm-water marine fishes, the optimal growth temperature is 20-28 ℃, the growth of the oplegnathus fasciatus is inhibited at the temperature lower than 13 ℃, the oplegnathus fasciatus is cold in northern winter, the oplegnathus fasciatus cannot naturally overwinter in a culture sea area, and the oplegnathus fasciatus needs to be transferred to an industrial workshop for overwinter conservation. Therefore, how to increase the growth rate of the oplegnathus punctatus, shorten the cultivation period on the market, reduce the production energy consumption in the cultivation process, and obtain high-quality products becomes a core problem concerned by the cultivation enterprises at present.

Disclosure of Invention

The invention aims to provide a spot sea relay culture method for oplegnathus punctatus, namely a method for industrially circulating water and carrying out net cage and fence relay culture on the oplegnathus punctatus.

The method for relay culture of oplegnathus punctatus in upland sea provided by the invention is characterized in that oplegnathus punctatus cultured in land-based industrial circulating water is transported to a net cage and/or a fence for culture by a live fish transport ship at the beginning of 6 months at the end of 5 months every year; and feeding the feed rich in glycine, lysine and methionine before the transfer;

further, feeding feed rich in glycine, lysine and methionine 10-15 days before transportation;

the feed rich in glycine, lysine and methionine is prepared by adding glycine, lysine and methionine into Sparus punctatus feed;

further, yeast capable of expressing the antibacterial peptide is added into the feed;

a specific amino acid sequence of the antibacterial peptide is as follows (SEQ ID NO: 1): LGLDFKFKEKKSIGFKEKHIQVRIFKDLNLRVKTEWH, respectively;

a specific sequence of the nucleotide fragment encoding the above antibacterial peptide is as follows (SEQ ID NO: 2):

CTGGGTCTGGATTTCAAATTCAAAGAAAAGAAGAGCATTGGTTTTAAAGAGAAGCATATCCAAGTCCGCATCTTCAAGGACCTGAACCTGCGCGTTAAAACCGAATGGCAC。

preferably, the feeding is stopped not less than 16 hours before the transportation of the oplegnathus punctatus;

further, the transported oplegnathus punctatus weighs not less than 90 g.

During transportation, the water temperature is kept at 19-25 ℃, and the dissolved oxygen of the water body is kept at 7mg/L or above.

According to the invention, the feed rich in glycine, lysine and methionine is fed to the oplegnathus maculatus before transportation, so that the stress of transportation to the oplegnathus maculatus is effectively reduced, the adaptation time of the factory-cultured oplegnathus maculatus to the natural ecological environment after transportation is shortened, and the normal ingestion of the oplegnathus maculatus is promoted, so that the growth can be recovered more quickly, and meanwhile, the yeast capable of expressing the antibacterial peptide is added into the feed, so that the non-specific immunity of the oplegnathus maculatus can be improved, and the stress resistance to the environment is enhanced.

Drawings

FIG. 1: photo images of the oplegnathus fasciatus cultured in land-based industrial circulating water;

FIG. 2: the activity of lysozyme and interferon in the blood serum of the oplegnathus punctatus and the phagocytic index diagram of macrophages; wherein (A) lysozyme (B) interferon (C) phagocytosis index;

FIG. 3: the liver antioxidant index map of the oplegnathus punctatus comprises (A) superoxide dismutase, (B) glutathione peroxidase and (C) catalase.

Detailed Description

The applicant adopts the method that fasting is carried out 18 hours before transportation in the process of transporting the oplegnathus fasciatus, and excrement residual feed in a temporary culture pond is removed every 6 hours during the fasting period; in the transportation process, vitamin C is added into the water body; wherein the vitamin C is added at a concentration of 10-100mg/L, and if the transport time is less than 12h, the transport density of Oplegnathus punctatus is not more than 100kg/m³A body of water; if the transportation time is 12-24 hr, the transported density of Oplegnathus punctatus is not more than 80kg/m³A body of water; if the transportation time exceeds 24h, the transportation density of the oplegnathus fasciatus does not exceed 65kg/m³A body of water; the dissolved oxygen of the water body is maintained at 7mg/L or more. After the transportation, the porgy is fed with the feed added with the bile acid, and after the feed added with the bile acid is fed for one week, the porgy is fed with the feed without the bile acid.

By the above method, although the mortality rate due to transport is significantly reduced; however, the ingestion rate of the oplegnathus punctatus is obviously influenced due to the change of the culture environment (from factory circulating water to deep and far sea nets and/or fences), and the ingestion rate is only 0.5% in the first week of transportation; this results in the inhibition of the growth of the transported oplegnathus fasciatus at the initial stage of the culture.

Through research on the amino acid composition of the liver of the oplegnathus punctatus before and after transportation, the contents of glycine, lysine and methionine in the liver of the oplegnathus punctatus are found to be obviously reduced compared with the contents of glycine, lysine and methionine in the liver of the oplegnathus punctatus one week after transportation before and after transportation. The present invention has been made on the basis of this finding.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1: pagrus punctatus in land-based industrial culture stage

1) Classification screening of oplegnathus punctatus in land-based industrial breeding stage

The volume of the water pool for cultivation is 40m³The depth of the pond is 1.5m, and the depth of water injection is about 1 m. The culture water is seawater filtered by three-level sand, the incubation water temperature is about 22 ℃, the culture water temperature of the oplegnathus fasciatus fry is 24-26 ℃, and the dissolved oxygen is largeAnd (3) configuring a dimming facility at 6mg/L with continuous micro-aeration, 24-28 salinity, 7.9-8.4 pH value and 500-800 lx illumination intensity. And after 20 days of age, carrying out dirt suction and bottom cleaning for 1 time every 4-5 days, continuously flowing water or discontinuously flowing water, wherein the daily flow is 12-18 times of the pond water, and selecting different culture densities (shown in table 1) according to the full length and the weight of the oplegnathus punctatus to obtain the optimal culture densities in different growth stages.

Table 1: survival rate and bait coefficient of oplegnathus fasciatus with different specifications under different culture densities

When the weight of the cultured oplegnathus fasciatus exceeds 90 grams, the culture density is kept at 60 tails/m³And preparing to transfer to a net cage and a fence for relay culture.

Example 2: transporting the Lutraria punctata cultured on land according to the existing method

1) Stopping feeding the oplegnathus fasciatus for not less than 16 hours before transportation, and preparing for transportation;

further, fasting is selected to be carried out 18 hours before transportation, and feces residue in the temporary rearing pond is removed every 6 hours during the fasting period;

2) in the transportation process, vitamin C is added into the water body; wherein the addition concentration of vitamin C is 10-100 mg/L;

wherein if the transportation time is less than 12h, the transportation density of the oplegnathus fasciatus is not more than 100kg/m³A body of water;

if the transportation time is 12-24 hr, the transported density of Oplegnathus punctatus is not more than 80kg/m³A body of water;

if the transportation time exceeds 24h, the transportation density of the oplegnathus fasciatus does not exceed 65kg/m³A body of water;

the dissolved oxygen of the water body is maintained at 7mg/L or more.

3) Feeding the porgy with the feed added with the bile acid after transportation, and feeding the porgy with the feed without the bile acid after one week;

wherein the feed added with bile acid is fed, and the feeding amount of the feed is 6 g/kg of body weight; the addition amount of bile acid is 2.0g/kg feed.

Through the measures, the oplegnathus maculatus is transported to a fence for culture, the transportation survival rate is 100%, the mortality rate is only 0.01% within 1 week after transportation, but the ingestion rate of the oplegnathus maculatus is not positive within the first week after transportation, the ingestion rate is only 0.5%, the ingestion of the oplegnathus maculatus is gradually recovered to be normal along with the prolonging of the culture period, the ingestion rate is 2.8% and the normal ingestion is gradually recovered within the 3 rd week after transportation. Therefore, although the transportation survival rate is guaranteed to be 100% by adopting the measures before, during and after the transportation, the ingestion movement of the oplegnathus fasciatus gradually returns to normal after 3 weeks of transportation, and the growth of the oplegnathus fasciatus is influenced in the early cultivation stage after the transportation and is almost in a stagnation state.

Example 3: analysis of amino acids in liver of oplegnathus fasciatus before and after transportation

The amino acid content in the liver was determined for the oplegnathus fasciatus cultured in the same culture pond of example 1 before and after the transport in the method of example 2 and one week of culture. Wherein 10 fish were selected for detection from non-transported and transported Oplegnathus fasciatus.

Freeze-drying liver tissue of the fish to be detected, and crushing the liver tissue by using a tissue homogenizer; the content of amino acid in liver is analyzed and determined by using an automatic amino acid analyzer. The measurement results are expressed as mean ± standard deviation, and the measurement data is analyzed by SPSS11.0 software.

The results showed that the contents of glycine ALA, lysine LYS and methionine MET in the liver were significantly reduced compared to the oplegnathus fasciatus one week after the transportation before the transportation (table 3); it is presumed that glycine ALA, lysine LYS and methionine MET are excessively consumed due to disturbance of metabolism of Oplegnathus punctatus induced by the transport process and stress reaction generated after transport to adapt to the natural environment.

Table 3: composition and content of partial amino acids in liver of oplegnathus fasciatus before and after transportation^*Indicating significant difference)

Example 4: feeding feed rich in glycine, lysine and methionine and transferring

1. Feeding of bait

Feeding the feed rich in glycine, lysine and methionine 2 weeks before the transfer, wherein the glycine, lysine and methionine are added to the granular feed for oplegnathus punctatus by spray drying, and the addition amount of the glycine, lysine and methionine is 0.1-0.5% of the weight of the feed respectively.

Meanwhile, in order to improve the resistance of the oplegnathus punctatus and the nonspecific immunity of the oplegnathus punctatus, saccharomycetes capable of expressing the antibacterial peptide are added into the feed.

The antibacterial peptide is obtained by screening from an inhibitory subtractive hybrid library, and is prepared by injecting bacteria into the abdominal cavity of the oplegnathus punctatus for immunization, extracting liver tissues of an experimental group and a control group respectively, and obtaining a library of differentially expressed genes after inhibitory subtractive hybridization.

Considering that natural antimicrobial peptides are generally polypeptides consisting of less than 50 amino acid residues, the molecular weight is about 2000-5000 daltons. Therefore, a cDNA fragment having a fragment size of 200bp or less is selected from a cDNA library obtained after inhibitory subtractive hybridization of oplegnathus punctatus, the amino acid sequence of the encoded polypeptide is inputted into an Antimicrobial Peptide Database (APD) for correlation analysis, and the nucleotide fragment having a similarity higher than 40% is subjected to further antimicrobial analysis. The final screening yielded the antimicrobial peptide whose amino acid sequence is as follows (SEQ ID NO: 1):

LGLDFKFKEKKSIGFKEKHIQVRIFKDLNLRVKTEWH；

the sequence of the encoding gene is as follows (SEQ ID NO: 2):

CTGGGTCTGGATTTCAAATTCAAAGAAAAGAAGAGCATTGGTTTTAAAGAGAAGCATATCCAAGTCCGCATCTTCAAGGACCTGAACCTGCGCGTTAAAACCGAATGGCAC。

the antibacterial property of the antibacterial peptide is detected by a trace broth dilution method, the Minimum Inhibitory Concentration (MIC) is carried out on commonly used fish pathogenic bacteria, and detected strains comprise vibrio anguillarum, edwardsiella tarda, aeromonas salmonicida and escherichia coli. The Minimum Inhibitory Concentration (MIC) results for the strains are shown in table 4.

Table 4: minimum inhibitory concentration table for pathogenic bacteria

As can be seen from the above results, the antimicrobial peptides used in the present invention have excellent antimicrobial effects.

2. Transport sparus punctatus

The feeding is stopped 2 days before the transportation, and the weight of the transported Oplegnathus punctatus is not less than 90 g. During transportation, the water temperature is kept at 19-25 ℃, and the dissolved oxygen of the water body is maintained above 7 mg/L. Wherein if the transportation time is less than 12h, the transportation density of the oplegnathus fasciatus is not more than 100kg/m³A body of water; if the transportation time is 12-24 hr, the transported density of Oplegnathus punctatus is not more than 80kg/m³A body of water; if the transportation time exceeds 24h, the transportation density of the oplegnathus fasciatus does not exceed 65kg/m³A body of water.

Example 5: detection of economic traits such as growth and resistance after transportation

Selecting 60,000 oxpoch 90g in 60,000 XUE in factory circulating water workshop in late 6 Yue of 2020, adding yeast rich in glycine, lysine and methionine and recombinantly expressing antibacterial peptide into oxpoch feed by the method of example 4, starting transportation after two weeks, fasting for 18 hours before transportation, selecting clear weather, and passing through live fish truck (transportation density 100 kg/m)³Water body, transportation time is 2 hours) to the wharf, and then the fish is transported by a professional live fish transport ship (transportation density is 100 kg/m)³Water, transport time 4 hours) to open sea breeding pens.

The swimming ingestion condition of the oplegnathus fasciatus is observed, the swimming and ingestion state of the oplegnathus fasciatus is obviously improved after 1 week, the ingestion rate reaches 1.0 percent within 1 week, the statistical transportation survival rate is 100 percent, the mortality rate is only 0.016 percent (death rate is 10 tails) within 1 week after transportation, the body weight is increased to 235 grams after 3 months of culture, and the mortality rate is 0.06 percent (death rate is 40 tails).

And the oplegnathus punctatus with the same specification and size is normally fed without yeast, glycine, lysine and methionine, is not fasted and is not fed in a limited manner by adopting a conventional transportation method, and the result shows that the transportation survival rate is 99.45 percent (death 332 tails) and the mortality rate in one week is 1.12 percent (death 672 tails), and the weight of the oplegnathus punctatus is increased to 198 g and the mortality rate is 0.35 percent (death 212 tails) after 3 months of culture. 1500 sparus punctatus of the same specification and size is cultured in an industrial circulating water workshop all the time, after 3 months of culture, the weight is increased to 179 g, and the death rate is 1.06% (16 tails died).

Meanwhile, the non-specific immune indexes (lysozyme, interferon and phagocytic index) and liver oxidation resistance indexes (superoxide dismutase, glutathione peroxidase and catalase) of the oplegnathus fasciatus are detected, and the results show that the activities of serum lysozyme, interferon and macrophage phagocytic index of the oplegnathus fasciatus are obviously higher than those of an unadditized group and an industrial culture circulating water culture group (figure 2) by adding saccharomycetes, glycine, lysine and methionine into the feed, and the activities of liver superoxide dismutase, glutathione peroxidase and catalase also show similar change trends. This indicates that by adopting the feeding strategy of the invention, the nonspecific immunity of the oplegnathus fasciatus is remarkably improved (figure 3), and the disease resistance is remarkably enhanced.