Application of lactic acid in breeding of artemia and a simple and healthy breeding method of artemia

Abstract

The application provides application of lactic acid in breeding of artemia. The lactic acid is directly added into the breeding water body by using silica or chitosan as a carrier. When the silica is used as the carrier, the use concentration of the lactic acid in the water body is 180-300 μM. When the chitosan is used as the carrier, the use concentration of the lactic acid in the water body is 230-600 μM. The inventors accidentally find that after the lactic acid is mixed with the silica or the chitosan and then added into the breeding water body, the pH of the water body does not decrease suddenly, so that an acid-base regulator does not need to be added, and the hatching and growth of the artemia are not affected. Meanwhile, by using the carrier, the local high concentration of the acid in the water body is realized, the bacteriostatic effect is obviously improved, and the use amount of the acid is reduced. On this basis, the application further provides a breeding method of artemia by using lactic acid as an alternative product. The artemia obtained by using the breeding method not only obviously improves the hatching rate and survival rate of the artemia, effectively reduces the number of vibrio carried by the artemia, but also up-regulates the expression of growth and immune related genes of the artemia, promotes the synchronous development and growth of the artemia, and achieves an unpredictable technical effect.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture technology and relates to a method for raising brine shrimp. Specifically, it relates to a method for using lactic acid to reduce the risk of brine shrimp carrying conditionally pathogenic bacteria, thereby achieving a simple, healthy, stable, and sustainable method for raising brine shrimp. Background Technology

[0002] Artemia, also known as brine shrimp, is a small, lower crustacean. Artemia is characterized by its small size, high nutritional value, strong environmental adaptability, rapid reproduction, and long-term storage capacity, making it an important biological feed for aquaculture. In terms of market demand, artemia has consistently been in short supply; high-density, healthy aquaculture of artemia has significant application prospects and substantial economic benefits. The non-selective filter-feeding nature of artemia often allows for the enrichment of different nutrients, improving its nutritional value. However, this also makes it prone to the accumulation of opportunistic pathogens, reducing hatching and survival rates, and causing sudden collapse of high-density aquaculture systems, leading to instability in artemia farming. Once artemia carries opportunistic pathogens, it can also infect the larvae of aquaculture fish, shrimp, and crabs through the food chain, causing seedling production failure. Therefore, effectively controlling the conditionally pathogenic bacteria carried by brine shrimp (especially pathogenic Vibrio, such as Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio campbellii) is crucial for ensuring the high-density sustainable farming of brine shrimp, as well as for the breeding of aquatic economic animals and the healthy farming of aquatic animals.

[0003] Antibiotics are commonly used in aquaculture to control opportunistic pathogens carried by brine shrimp. However, the use of antibiotics is not only limited in its effectiveness but also promotes the emergence of drug-resistant pathogens, which are then transmitted through the food chain, posing a significant threat to human health and the ecological environment. Currently, acidifiers (such as formic acid and acetic acid), microecological preparations (such as marine Bacillus), and plant extracts (such as essential oils) are used as alternatives to antibiotics in brine shrimp farming. Among them, acidifiers refer to a general term for organic acids that can be used as additives, belonging to non-nutritive feed additives. However, the antibacterial effect of acids weakens as the pH increases, which contradicts the fact that the natural environment in which brine shrimp live is neutral to slightly alkaline. Existing studies have shown that the optimal hatching pH for brine shrimp is 7.5-8.5. If the pH is <7, it will lead to softening of the eggshell, embryonic malformation, and a significant reduction in hatching rate. Although adults can adapt to a wide pH range and can survive at pH 7.0-9.5, the optimal growth pH is 7.5-8.5. Furthermore, sudden pH changes can trigger stress responses in artichokes (such as abnormal molting, decreased immunity, and increased susceptibility to infection). Immanuel et al. studied the effects of caprylic acid, acetic acid, propionic acid, and butyric acid on the survival rate of sterile artichokes artificially infected with Vibrio harveyi and Vibrio parahaemolyticus under experimental conditions. Adding caprylic acid (10 mM) to the culture water significantly reduced the mortality rate of artichokes infected with Vibrio harveyi by 16.30% and those infected with Vibrio parahaemolyticus by 20.61% (P < 0.05). Using seawater supplemented with acetic acid (20 mM), propionic acid (20 mM), and butyric acid (20 mM) to culture artichoke nauplii infected with Vibrio parahaemolyticus, the mortality rates were reduced by 16.7%, 11.7%, and 10.0%, respectively, compared to the control group. However, although the mortality rate of artichokes decreased, the high concentrations of the aforementioned acids resulted in excessively low pH levels in the water, necessitating the addition of acid-base regulators to adjust the pH to 7 to meet the environmental requirements of the artichokes. In actual aquaculture production, this significantly increases costs and operational steps, hindering the growth and industrial application of brine shrimp. Furthermore, as mentioned earlier, this study focused on artificially infected, sterile brine shrimp, which carry a simpler microbial community compared to actual aquaculture systems.

[0004] Lactic acid is a carboxylic acid containing a hydroxyl group, with a molecular weight of 90.08 (g / mol) and an acidity coefficient of 3.86, between that of formic acid and acetic acid. It usually exists in liquid form and is readily soluble in water. However, directly applying lactic acid to aquaculture water presents a series of problems, including easy dissociation, irritation, and sensitivity to alkaline environments. Furthermore, high hardness water, alkalinity, and organic matter can neutralize organic acids, thus weakening its antibacterial effects. Increasing the lactic acid concentration, however, can cause a sharp drop in water pH, exceeding the tolerance range of artichokes, inhibiting their hatching and growth, and further increasing costs and toxicity risks.

[0005] Due to the aforementioned technical issues, there are currently no reports of lactic acid being used in brine shrimp farming. Summary of the Invention

[0006] To address the problems of cumbersome procedures and high costs associated with using organic acids as antibiotic alternatives in existing brine shrimp farming techniques, this invention first provides the application of lactic acid in brine shrimp farming. This application achieves both the antibacterial effect of lactic acid and stable water pH without the addition of acid-base regulators, overcoming the technical bias of existing techniques that cause a sharp drop in water pH when increasing lactic acid concentration. Based on this, this application also provides a method for farming brine shrimp using lactic acid as an antibiotic alternative. Brine shrimp obtained using this method not only significantly improve the hatching and survival rates of brine shrimp and effectively reduce the number of Vibrio bacteria carried by the brine shrimp, but also upregulate the expression of growth and immune-related genes, promoting synchronous development and growth of the brine shrimp and achieving unexpected technical effects.

[0007] The technical solution of this invention:

[0008] The application of lactic acid in brine shrimp farming involves directly adding the lactic acid to the farming water using silica or chitosan as a carrier. When silica is used as the carrier, the concentration of lactic acid in the water is 180-300 μM; when chitosan is used as the carrier, the concentration is 230-600 μM. The inventors unexpectedly discovered that mixing lactic acid with the aforementioned silica or chitosan before adding it to the farming water does not cause a sudden drop in pH, thus eliminating the need for acid-base regulators and not affecting the hatching and growth of brine shrimp. Simultaneously, the use of a carrier achieves a locally high concentration of acid in the water, significantly enhancing the antibacterial effect while reducing the amount of acid used. Therefore, the application described in this application achieves both the antibacterial effect of lactic acid and stable water pH, overcoming the technical bias of prior art where increasing the lactic acid concentration to achieve antibacterial effect leads to a sudden drop in water pH.

[0009] Preferably, when the lactic acid is supported by silica, the weight-to-volume ratio of SiO2 to lactic acid is (0.5~3):1 (g:ml), and the particle size of the silica is 10-40 μm; when the lactic acid is supported by chitosan, the weight-to-volume ratio of chitosan to lactic acid is (0.01~0.1):1 (g:ml), and the degree of deacetylation of the chitosan is 75-88%, with a molecular weight of 100-180 kDa. Previous studies have shown that the silanol groups (Si-OH) on the surface of SiO2 can interact with the carboxylic acid groups (-COOH or -COO-) of organic acids through hydrogen bonding, and chitosan can interact through protonated amino groups (-NH3). + ) and the carboxyl ion (-COO) of organic acids -This process forms ionic complexes, thereby controlling the release rate of organic acids, maintaining a high local concentration of acids, prolonging the action time of organic acids, and stabilizing the pH of the water. However, excessively high SiO2 or chitosan concentrations can affect the hatching rate of Artemia eggs and the survival rate of nauplii, while excessively low SiO2 or chitosan concentrations may not be able to stabilize the pH of the water.

[0010] Preferably, the density of Artemia eggs in the culture water does not exceed 1000 eggs / mL. This wide range of Artemia egg densities makes it suitable not only for experimental conditions but also for large-scale practical culture, thus possessing significant practical application value.

[0011] A simple and healthy method for cultivating artichokes includes the following steps:

[0012] (1) Mix carrier SiO2 or chitosan with lactic acid in a uniform manner according to the weight-volume ratio to obtain a lactic acid product in the form of emulsion or powder, wherein the weight-volume ratio of SiO2 to lactic acid is (0.5~3):1 (g:ml); the weight-volume ratio of chitosan to lactic acid is (0.01~0.1):1 (g:ml).

[0013] (2) Add the lactic acid product described in step (1) to the water used for the hatching and cultivation of brine shrimp, so that the concentration of lactic acid in the water reaches the required level, thereby obtaining lactic acid aquaculture water. Wherein, when the lactic acid is carried by silica, the concentration of lactic acid in the water is 180-300 μM, and the particle size of the silica is 10-40 μm; when the lactic acid is carried by chitosan, the concentration of lactic acid in the water is 230-600 μM, and the degree of deacetylation of the chitosan is 75-88%, and the molecular weight is 100-180 kDa.

[0014] This step slows down the release rate of organic acids through the interaction between the carrier and lactic acid, maintains a high local concentration of acids (effective antibacterial effect), prolongs the action time and improves stability, avoids a sharp drop in the pH value of the water, and keeps the pH value of the water stable within the range of 7.5-8.5, which is suitable for the survival of Artemia.

[0015] (3) Artemia eggs are introduced into the lactic acid culture water body described in step (2), and incubated at 28-30℃ for 18-24 h. Basic feed is provided under continuous light conditions to obtain Artemia. The density of Artemia eggs in the water body is no more than 1000 eggs / mL; the continuous light conditions are 1000-2500 Lux; the basic feed is Chlorella, yeast powder, Spirulina powder, rice bran, soybean meal, or artificial microparticle feed.

[0016] The brine shrimp eggs used in this step are commercially farmed brine shrimp eggs, unlike the sterile brine shrimp eggs used in the previous studies. Therefore, they often carry opportunistic pathogens, such as Vibrio spp. Through the aforementioned farming process, not only was the number of opportunistic pathogens carried by the brine shrimp significantly reduced, but the diversity of the carried microbiota and the relative abundance of beneficial bacteria were also increased. This not only improved the health of the brine shrimp themselves and reduced mortality due to Vibrio infection, but also optimized the entire farming microecological environment, reduced the risk of disease in aquatic animals, and reduced the use of antibiotics and other drugs, aligning with the development trend of green and healthy aquaculture. The inventors believe that this is because lactic acid itself has antibacterial activity, and the slow-release effect of the carrier and the local high concentration allow them to continuously and effectively act on the brine shrimp's living environment and on its surface and inside its body for a relatively long period.

[0017] Furthermore, the brine shrimp cultured using this method exhibited significantly improved synchronous development and growth rates compared to conventional methods, resulting in unexpected technological advancements. Transcriptomics sequencing analysis and qRT-PCR verification by the inventors suggest that lactic acid, through the sustained-release effect of the carrier system, regulates the expression of growth- and immune-related genes in brine shrimp, comprehensively promoting their growth, development, and survival rate. This is a first-time discovery by the inventors, providing a theoretical basis for further optimizing brine shrimp culture technology. It not only shortens the culture cycle but also ensures high-density, sustainable, and healthy brine shrimp culture, possessing significant practical application value.

[0018] An application of brine shrimp in aquaculture, wherein the brine shrimp are obtained using the aforementioned cultivation method. Compared with existing technologies, the brine shrimp obtained using the aforementioned cultivation method not only carry a reduced number of pathogenic bacteria but also increase the diversity index of the carried microbial community and the stability of the aquaculture microecological environment. Therefore, applying the brine shrimp to aquaculture reduces the risk of disease in farmed animals while reducing antibiotic use, which aligns with the development trend of green and healthy aquaculture. Furthermore, the brine shrimp obtained using the aforementioned cultivation method not only have significantly improved hatching and survival rates but also, due to their synchronous development and healthier carried microbial community, become a high-quality live feed for aquaculture.

[0019] The beneficial effects of this invention are:

[0020] (1) This application firstly provides the application of lactic acid in brine shrimp farming. The application achieves both the antibacterial effect of lactic acid and the stability of water pH without the addition of acid-base regulators, overcoming the technical problems in the prior art where increasing the concentration of lactic acid leads to a sudden drop in water pH, and the concentration is too low to produce an antibacterial effect.

[0021] (2) This application provides a method for raising Artemia using lactic acid as an antibiotic alternative. The method significantly improves the hatching and survival rates of Artemia and effectively reduces the number of Vibrio bacteria carried by Artemia. This provides more healthy Artemia larvae for aquaculture, ensures the production of seedlings for aquatic economic animals, and improves the efficiency of aquaculture from the source.

[0022] (3) The breeding method described in this application promotes the synchronous growth and development of brine shrimp by regulating the expression of genes related to growth and immunity, enhances the immunity of brine shrimp, and ensures the healthy and sustainable breeding of brine shrimp. Compared with the existing technology that only utilizes the antibacterial properties of organic acids, the breeding method described achieves unpredictable technical effects and provides a theoretical basis for further optimizing brine shrimp breeding technology. Attached Figure Description

[0023] Appendix Figure 1 The effect of lactic acid concentration in aquaculture water (without carrier) on the hatching rate and survival rate of Artemia worms;

[0024] Appendix Figure 2 The lactic acid emulsions prepared using SiO2 as a carrier in Examples 1-3 of this application;

[0025] Appendix Figure 3 The powder prepared by using chitosan as a carrier for lactic acid in Examples 4-6 of this application;

[0026] Appendix Figure 4 The relative abundance of bacterial species carried by Artemia spp. in Example 7 is shown in the SiO2-based lactic acid group (LS), the chitosan-based lactic acid group (LC), and the blank control group.

[0027] Appendix Figure 5 The effect of the lactic acid group (LS) with SiO2 as a carrier and the lactic acid group (LC) with chitosan as a carrier on the number of Vibrio carried by Artemia in Example 7 (day 7).

[0028] Appendix Figure 6 This is a comparison of the developmental synchronicity of the lactic acid group (LS) with SiO2 as the carrier and the blank control group 24 hours after hatching in Example 9;

[0029] Appendix Figure 7 This is a KEGG enrichment analysis diagram of differentially expressed genes in the lactic acid group (LS) and the control group of Artemia using SiO2 as a carrier in Example 10.

[0030] Appendix Figure 8 This is a KEGG enrichment analysis diagram of differentially expressed genes in the lactic acid group (LC) and the control group of Artemia salina using chitosan as a carrier in Example 10.

[0031] Appendix Figure 9The results of qRT-PCR for differentially expressed genes in Artemia salina in Example 10 are shown (A. Lactic acid group (LS) with SiO2 as a carrier and blank control group, B. Lactic acid group (LC) with chitosan as a carrier and blank control group). Detailed Implementation

[0032] The invention will be further described below with reference to the embodiments. The Artemia eggs (80% hatching rate), food-grade SiO2, chitosan, and lactic acid used in the embodiments were all commercially available. The pH of the water used for Artemia hatching and culture was 8.23.

[0033] 1. The effect of lactic acid on the hatching and survival of artichokes without a carrier.

[0034] To determine the safe concentration of lactic acid for improving the hatching and survival rates of Artemia, the following experiment was conducted (experimental group and control group).

[0035] The experimental groups were treated with different concentrations of lactic acid (0, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000 μM) in sterile seawater; the control group received no treatment and underwent Artemia hatching and cultivation. Specifically, Artemia eggs were hatched at a density of 30 eggs / mL in 5 mL of sterile seawater for 24 h, with the culture water temperature maintained at 28-30 °C and continuous illumination (2000 Lux). Chlorella was fed twice daily at 7:00 AM and 7:00 PM. The total number of Artemia eggs (H), the total number of hatched Artemia (h), and the total number of nauplius surviving larvae (s) after 48 h were recorded. Each group was divided into three replicates. See below for detailed results. Figure 3 .

[0036] like Figure 1 As shown, lactic acid concentration below 80 μM had no significant effect on the hatching rate of Artemia eggs (P > 0.05) and no significant effect on the survival rate of Artemia nauplii (P > 0.05). With increasing lactic acid concentration, both the hatching rate of Artemia eggs and the survival rate of nauplii gradually decreased. The LC50 of the effect of lactic acid on the hatching rate of Artemia eggs was calculated based on the number of dead Artemia eggs and nauplii. 50 Values ​​and LC for nauplius survival 50 The values ​​were 415.77 μM and 350.53 μM, respectively.

[0037] According to Turubell's safe concentration calculation formula:

[0038]

[0039] The safe concentration of lactic acid for Artemia was found to be 74.75 μM.

[0040] Example 1: Hatching and rearing of Artemia (lactic acid-SiO2 as an antibiotic alternative)

[0041] A simple and healthy method for cultivating artichokes includes the following steps:

[0042] (1) Preparation of lactic acid products: SiO2 and lactic acid are mixed evenly according to the weight-volume ratio to obtain lactic acid products in the form of emulsions (e.g. Figure 2 (As shown); the weight-to-volume ratio of SiO2 to lactic acid is 0.6:1 (g:ml).

[0043] (2) Preparation of lactic acid aquaculture water: The lactic acid product described in step (1) is added to the water used for the hatching and aquaculture of brine shrimp to achieve the required concentration of lactic acid in the aquaculture water, thereby obtaining lactic acid aquaculture water. The concentration of lactic acid in the water is 180 μM, and the particle size of the silica is 10 μm.

[0044] (3) Artemia hatching and cultivation: Artemia eggs are placed in the lactic acid culture water body described in step (2) and hatched at 28-30℃ for a period of time. Basic feed is provided under continuous light conditions to obtain Artemia. The hatching time is 18 hours, the density of Artemia eggs in the water body is 100 eggs / mL, the continuous light condition is 1000 Lux, and the basic feed is Chlorella, which is fed twice a day at 7:00 and 19:00.

[0045] (4) Detection of brine shrimp hatching rate, survival rate and body length:

[0046] Artemia hatching rate and survival rate: Hatching was completed according to step (3), and the total number of artemia eggs (H), the total number of hatched artemia (h), and the total number of nauplii surviving at 48 h (s) were recorded. Three parallels were set up for each group. The hatching rate was expressed as the percentage of the total number of hatched artemia (h) to the total number of artemia eggs (H), and the survival rate was expressed as the percentage of the total number of nauplii surviving at 48 h to the total number of artemia eggs (H), as shown in the following formula:

[0047]

[0048]

[0049] Note: h is the total number of hatched artichokes; H is the total number of artichoke eggs; s is the total number of nauplii surviving after 48 hours.

[0050] Body length measurement: Starting from the hatching of the artichokes, 10 artichokes were randomly selected from each experimental group and control group every day. The body length of the artichokes was measured using a stereomicroscope once a day. The body length was the distance from the front end of the head to the bottom of the tail fork. The body length of the artichokes was recorded and the average value was calculated.

[0051] Example 2: Hatching and rearing of Artemia (lactic acid-SiO2 as an antibiotic alternative)

[0052] Unlike Example 1, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0053] (1) Preparation of lactic acid products: The weight-volume ratio of SiO2 to lactic acid is 1.5:1 (g:ml).

[0054] (2) Preparation of lactic acid aquaculture water: The concentration of lactic acid in the water is 250 μM, and the particle size of the silica is 20 μm.

[0055] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 1000 eggs / mL; the continuous light condition is 2000 Lux; the basic feed is spirulina powder, which is fed twice a day at 7:00 and 19:00.

[0056] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0057] Example 3: Hatching and rearing of Artemia (lactic acid-SiO2 as an antibiotic alternative)

[0058] Unlike Example 1, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0059] (1) Preparation of lactic acid products: The weight-volume ratio of SiO2 to lactic acid is 2.5:1 (g:ml).

[0060] (2) Preparation of lactic acid aquaculture water: The concentration of lactic acid in the water is 300 μM, and the particle size of the silica is 40 μm.

[0061] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 500 eggs / mL; the continuous light condition is 2500 Lux; the basic feed is yeast powder, which is fed twice a day at 7:00 and 19:00.

[0062] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0063] Comparative Example 1: Hatching and rearing of Artemia (lactic acid is an antibiotic alternative)

[0064] Unlike Example 2, no SiO2 was added.

[0065] Example 4: Hatching and rearing of Artemia (lactic acid-chitosan as an antibiotic alternative)

[0066] A simple and healthy method for cultivating artichokes includes the following steps:

[0067] (1) Preparation of lactic acid products: Chitosan and lactic acid are mixed evenly according to the weight-volume ratio to obtain lactic acid products in powder form (e.g. Figure 3 (As shown); the weight-to-volume ratio of chitosan to lactic acid is 0.01:1 (g:ml).

[0068] (2) Preparation of lactic acid aquaculture water: The lactic acid product described in step (1) is added to the water used for the hatching and aquaculture of brine shrimp to achieve the required concentration of lactic acid in the aquaculture water, thus obtaining lactic acid aquaculture water. The concentration of lactic acid used in the water is 250 μM, and the degree of deacetylation of the chitosan is 75%, with a molecular weight of 180 kDa.

[0069] (3) Artemia hatching and cultivation: Artemia eggs are placed in the lactic acid culture water body described in step (2) and hatched at 28-30℃ for a period of time. Basic feed is provided under continuous light conditions to obtain Artemia. The hatching time is 18 hours, the density of Artemia eggs in the water body is 100 eggs / mL, the continuous light condition is 1000 Lux, and the basic feed is Chlorella, which is fed twice a day at 7:00 and 19:00.

[0070] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0071] Example 5: Hatching and rearing of Artemia (lactic acid-chitosan as an antibiotic alternative)

[0072] Unlike Example 4, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0073] (1) Preparation of lactic acid products: The weight-volume ratio of chitosan to lactic acid is 0.05:1 (g:ml).

[0074] (2) Preparation of lactic acid aquaculture water: The concentration of lactic acid used in the water is 500 μM, the degree of deacetylation of chitosan is 80%, and the molecular weight is 100 kDa.

[0075] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 1000 eggs / mL; the continuous light condition is 2000 Lux; the basic feed is spirulina powder, which is fed twice a day at 7:00 and 19:00.

[0076] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0077] Example 6: Hatching and rearing of Artemia (lactic acid-chitosan as an antibiotic alternative)

[0078] Unlike Example 4, a simple and healthy method for cultivating brine shrimp includes the following steps:

[0079] (1) Preparation of lactic acid products: The weight-volume ratio of chitosan to lactic acid is 0.1:1 (g:ml).

[0080] (2) Preparation of lactic acid aquaculture water: The concentration of lactic acid used in the water is 600 μM, the degree of deacetylation of chitosan is 88%, and the molecular weight is 120 kDa.

[0081] (3) Artemia hatching and culture: The hatching time is 24 hours, the density of Artemia eggs in the water is 500 eggs / mL; the continuous light condition is 2500 Lux; the basic feed is yeast powder, which is fed twice a day at 7:00 and 19:00.

[0082] (4) Detection of brine shrimp hatching rate, survival rate and body length: Same as in Example 1.

[0083] Comparative Example 2: Hatching and rearing of Artemia (lactic acid is an antibiotic alternative)

[0084] Unlike Example 5, chitosan was not added.

[0085] Comparative Example 3: Hatching and rearing of brine shrimp (blank control group)

[0086] Unlike Example 5, no lactic acid, SiO2, or chitosan were added.

[0087] Table 1. Hatching rate, survival rate, and body length of cultured artichokes in Examples 1-6 and Comparative Examples 1-2

[0088]

[0089] As shown in Table 1, compared with Example 3, the blank control (without lactic acid and carrier addition) had a culture water pH of 8.23, an Artemia hatching rate of 81% and a 48-hour survival rate of 78%, and an average body length of 3.46 mm on the 7th day.

[0090] Based on the Turubell safe concentration calculation formula, the safe concentration (74.75 μM) of lactic acid for the hatching rate and survival rate of Artemia larvae was obtained. In Examples 1-3, which used lactic acid-SiO2 as an antibiotic alternative, the initial pH of the culture water was 8.23. After adding lactic acid-SiO2 at concentrations exceeding the safe concentration (180-300 μM), the final pH of the culture water only decreased slightly, remaining between 7.58 and 7.86. The hatching rate of Artemia larvae eggs was 80-82%, the 48-hour survival rate was 77-78%, and the average body length on day 7 was 3.74-3.83 mm. In contrast, in Comparative Example 1, which used only lactic acid as an antibiotic alternative, 250 μM lactic acid lowered the final pH of the culture water to 6.86, significantly impacting the hatching rate and larval survival rate of Artemia larvae. The hatching rate significantly decreased to 53%, the 48-hour survival rate was 48%, and the survival rate was even worse in later stages. The hatching and survival rates of Artemia worms in Examples 1-3 were significantly higher than those in Comparative Example 1, but there was no significant difference compared to Comparative Example 3. The Artemia worm body length in Examples 1-3 was significantly promoted compared to Comparative Example 3.

[0091] In Examples 4-6, which used lactic acid-chitosan as an antibiotic alternative, the initial pH of the culture water was 8.23. After adding lactic acid-chitosan at concentrations far exceeding the safe usage level (250-600 μM), the final pH of the culture water only decreased slightly, remaining between 7.69 and 7.94. The hatching rate of Artemia eggs was 77-79%, the survival rate was 71-74%, and the average body length on day 7 was 3.77-3.99 mm. In contrast, in Comparative Example 2, which used only lactic acid as an antibiotic alternative, 500 μM lactic acid lowered the final pH of the culture water to 6.72, affecting the hatching rate of Artemia eggs and the survival of Artemia larvae, resulting in a hatching rate of 50%, a 48-hour survival rate of 44%, and poor survival rates in the later stages. The hatching rate and survival rate of Artemia in Examples 4-6 were significantly higher than those in Comparative Example 2, but slightly lower than those in Comparative Example 3. The increase in body length of Artemia in Examples 4-6 was more significant than that in Comparative Example 3.

[0092] Therefore, compared with the carrier-free system, (1) after adding 180-600 μM lactic acid-carrier to the aquaculture water in Examples 1-6 of this application, the final pH was 7.58-7.94, and the pH of the aquaculture water remained stable, which was suitable for the growth and reproduction of brine shrimp; (2) the hatching rate and survival rate of brine shrimp obtained by the aquaculture methods in Examples 1-6 of this application were significantly improved (P < 0.05); (3) compared with the blank control system (without lactic acid and carrier addition), without significantly affecting the hatching rate and survival rate of brine shrimp, it not only promoted the growth of brine shrimp (significantly increased body length), but also increased the safe concentration of lactic acid (i.e., improved the antibacterial effect), achieving an unexpected technical effect. This shows that the lactic acid-SiO2 and lactic acid-chitosan are antibiotic alternatives. Without adding acid-base regulators, they overcome the technical problem in the prior art that increasing the concentration of lactic acid leads to a sudden drop in the pH of the water, which affects the hatching and survival of brine shrimp, and achieves the unexpected technical effect of promoting the growth and development of brine shrimp.

[0093] Example 7: Analysis of the microbial community carried by cultured brine shrimp

[0094] This embodiment performs high-throughput sequencing analysis based on 16S rRNA on the bacterial communities carried by cultured Artemia fowl in Examples 1-6, and the sequencing results are consistent with the actual results. The following detailed explanation uses Examples 2 (LS group) and Example 5 (LC group) as examples, combined with the blank control group (Control, without added lactic acid and vector), as examples:

[0095] (1) Sample preparation

[0096] Starting from the hatching of the artichokes, samples were taken on day 1 (nauplius stage), day 3 (pseudo-adult stage), and day 7 (adult stage). From each group's three replicates, 200 artichokes were randomly selected, mixed, repeatedly rinsed with ultrapure water, placed in 1.5 mL EP tubes, flash-frozen in liquid nitrogen for 30 minutes, and then stored at -80 °C for later use. After all samples were collected, Shanghai Meiji Biotechnology Co., Ltd. performed 16S rRNA-based microbial community analysis of the artichokes.

[0097] (2) Data Analysis

[0098] Pair-end reads obtained from Illumina sequencing were spliced ​​together based on overlap, and sequence quality was controlled and filtered. After sample differentiation, OTU (Operational Taxonomic Unit) clustering and taxonomic analysis were performed. Bioinformatics statistical analysis was typically conducted on OTUs with a similarity level of 97%. Based on taxonomic information, statistical analysis of community structure was performed at each taxonomic level.

[0099] Alpha diversity analysis was performed based on the high-throughput sequencing results of each group of Artemia samples, resulting in a diversity index statistical table (Table 2). The Chao, Ace, and Sob indices reflect the number of species in the samples, but do not consider the abundance of each species; the Shannon and Simpson indices reflect the influence of species abundance and evenness on the diversity of the samples. That is, under the same species abundance, the greater the evenness of each species in the sample, the greater its diversity is considered.

[0100] (3) The dilution plating method was used to verify the effect of lactic acid-carrier on the number of Vibrio carried by Artemia esculenta.

[0101] On day 7 (adult stage), 30 Artemia worms were collected from each of Example 2 (LS group), Example 5 (LC group), and the blank control group (Control, without lactic acid and carrier). In a clean bench, the Artemia worms were repeatedly rinsed with ultrapure water, and then 1 mL of physiological saline was added to a glass homogenizer for tissue homogenization until the Artemia tissue was no longer visible. The homogenized tissue solution was diluted 10 times and spread on TCBS plates. The plates were incubated at 32 °C for 2 days, and the morphological characteristics of the bacteria were observed and counted.

[0102] Lactic acid with SiO2 as a carrier (LS group) and lactic acid with chitosan as a carrier (LC group) were added to the brine shrimp culture water for brine shrimp culture. The relative abundance of bacterial community composition at the genus level of each group during the nauplius larval stage (day 1), pseudo-adult stage (day 3), and adult stage (day 7) of brine shrimp was analyzed. See details. Figure 4 .like Figure 4 As shown, the dominant bacterial groups with the highest relative abundance were analyzed. It was found that during the nauplius stage, the dominant bacterial groups carried by Artemia in the control group were *Exiguobacterium* and *Acinetobacter*, with relative abundances of 52.2% and 12.5%, respectively; those in the LS group were *Acinetobacter* and *Marinobacter*, with relative abundances of 14.4% and 7.6%, respectively; and those in the LC group were *Psychrobacter* and *Exiguobacterium*, with relative abundances of 52.6% and 37.3%, respectively. Vibrio was not observed as a dominant bacterial group in any of the three groups during the nauplius stage.

[0103] During the pseudo-adult stage, the dominant bacterial species carried by Artemia in the control group were *Psychrobacter* and *Microbacterium*, with relative abundances of 79.43% and 6.4%, respectively; those carried by Artemia in the LS group were *Marinebacter* and *Idiomarina*, with relative abundances of 25.4% and 4.3%, respectively; and those carried by Artemia in the LC group were *Pseudoalteromonas* and *Psychrobacter*, with relative abundances of 15.2% and 13.1%, respectively. Vibrio was not observed as a dominant bacterial species in any of the three groups during the pseudo-adult stage.

[0104] However, during the adult stage, Vibrio was abundant in both the blank control group and the LC group, becoming the dominant bacterial species. The dominant bacteria in the blank control group were Vibrio and Psychrophilic Bacillus, with relative abundances of 56.5% and 21.7%, respectively; the dominant bacteria in the LC group were Vibrio and Microbacterium, with relative abundances of 64.6% and 21.9%, respectively; while the dominant bacteria in the LS group were Marinebacterium and Microbacterium, with relative abundances of 8.9% and 5.1%, respectively, and Vibrio's relative abundance was only 0.28%. Previous studies have reported that Microbacterium, Psychrophilic Bacillus, and Marinebacterium are potential probiotics, and their increase can help enhance the gut health of farmed animals, improve their immunity and disease resistance.

[0105] Table 2. Alpha diversity analysis of the microbial communities carried by Artemia spp. at different growth stages in LS, LC and blank control groups.

[0106]

[0107] Table 2 shows that the estimated values ​​of Sobs (195, 102, 114), Chao (198.6, 106.4, 133.43), and Ace (197.5, 109.79, 126.43) indices of the Artemia larvae in the LS group at each growth stage were significantly higher than those in the LC group and the blank control group, indicating that lactic acid carried by SiO2 resulted in a richer variety of bacterial species carried by the Artemia larvae. The Shannon index (2.47, 2.49, 2.58) of the Artemia larvae in the LS group at each growth stage was higher than that in the LC group and the blank control group, indicating a better evenness of species distribution in the LS group; and its Simpson index (0.19, 0.17, 0.13) was lower than that in the LC group and the blank control group, indicating fewer dominant species and higher bacterial diversity. Compared with the LC group and the blank control group, the culture micro-ecosystem of the LS group was more stable and healthier.

[0108] Furthermore, the effect of lactic acid on the number of Vibrio carried by Artemia on day 7 was verified using the dilution plating method. See details below. Figure 5 .like Figure 5 The results showed that the number of Vibrio bacteria carried by Artemia spp. in group LS was 1.8 × 10⁻⁶.2 The number of Vibrio filaments (CFU / tail) in the LC group was significantly lower than that in the control group (P < 0.05), but the number of Vibrio filaments in the LC group was 3.27 × 10⁻⁶. 5 CFU / tail) compared to the control group (4.03×10 5 There was no significant difference in CFU / tail (P>0.05). The plating results were consistent with the high-throughput sequencing results. On day 7, the survival rate of Artemia in the LS group (78±5%) was significantly higher than that in the control group (68±8%) and the LC group (71±5%).

[0109] In summary, in this application, lactic acid with SiO2 as a carrier can significantly improve the hatching rate and survival rate of artichokes and effectively control the Vibrio carried by artichokes, and is superior to lactic acid with chitosan as a carrier.

[0110] Example 8: Anti-Vibrio Infection Experiment

[0111] Data from Examples 1-6 and Comparative Examples 1-2 show that using lactic acid-SiO2 and lactic acid-chitosan as antibiotic alternatives in Artemia culturing significantly improved the hatching rate, survival rate, and body length of the Artemia worms. Analysis of the microbial community carried by the cultured Artemia worms in Example 7 showed that lactic acid-SiO2 significantly reduced the number of conditionally pathogenic bacteria carried by the Artemia worms, and the carried microbial community was more diverse, with high distribution evenness and diversity index, indicating a more stable and healthy culture.

[0112] Furthermore, an artesian challenge experiment was conducted using Vibrio campbelli BB120 as the pathogen to verify the antibacterial effects of lactic acid-SiO2 and lactic acid-chitosan on the pathogenic Vibrio. The following details the use of Examples 2 (LS group) and 5 (LC group) as examples, where Vibrio campbelli BB120 was added during artesian hatching, combined with the Vibrio group (only Vibrio campbelli BB120 added) and the blank control group (no antibiotic alternative and Vibrio campbelli BB120 added).

[0113] Three experimental groups were established: Example 2 (lactic acid-SiO2 + Vibrio campbellii BB120), Example 5 (lactic acid-chitosan + Vibrio campbellii BB120), and a Vibrio group (only Vibrio campbellii BB120 was added). During the hatching of Artemia larvae eggs, Vibrio campbellii BB120 was added to the culture water of each experimental group at a concentration of 1×10⁻⁶. 7Challenge with a final concentration of cell / mL, while the blank control group received no treatment. The total number of Artemia eggs (H), the total number of hatched Artemia (h), and the total number of nauplius surviving at 48 h (s) were recorded. Each group had three replicates. Hatching rate was expressed as the percentage of hatched Artemia (h) to the total number of Artemia eggs (H), and survival rate was expressed as the percentage of surviving nauplius (s) to the total number of Artemia eggs (H), calculated using the same formula as in Example 1.

[0114] As shown in Table 3, at 1×10 7 Under challenge with Vibrio campeosa BB120 at cell / mL, the hatching rate and nauplius survival rate of Artemia eggs with added lactic acid-SiO2 or lactic acid-chitosan were significantly higher than those in the Vibrio group (P < 0.05), indicating that lactic acid-carrier can significantly reduce the adverse effects of Vibrio on Artemia hatching and growth. Furthermore, in the LS group with added lactic acid-SiO2, there was no significant difference in Artemia hatching rate and nauplius survival rate compared to the blank control group (P > 0.05), with lactic acid using chitosan as the carrier showing the second-best effect. These experimental results are consistent with those of Example 7.

[0115] Table 3. Effects of lactic acid-carrier on hatching rate and survival rate of Artemia under Vibrio campestris challenge conditions.

[0116]

[0117] Note: Different letters indicate significant differences (P < 0.05), while the same letter indicates no significant differences (P > 0.05).

[0118] Example 9: Measurement of Artemia growth rate and developmental synchronicity

[0119] Based on the data from Examples 1-6 and Comparative Examples 1-2, it is evident that using lactic acid-SiO2 and lactic acid-chitosan as antibiotic alternatives significantly increased the body length of Artemia larvae during cultivation. Using Examples 2 (LS group) and 5 (LC group) as examples, the inventors further investigated the effects on the growth rate of Artemia larvae in conjunction with a blank control group. The specific procedures are as follows:

[0120] Ten artichokes were randomly selected from each experimental group and control group. The body length of the artichokes was measured using a stereomicroscope once daily. The body length was measured as the distance from the front of the head to the base of the tail fork. The body length was recorded, and the average, average daily growth rate (ADG, mm / d), and specific growth rate (SGR, %) were calculated. The formulas for calculating the average daily growth rate and specific growth rate are as follows:

[0121]

[0122]

[0123] Note: ADG is the average daily growth rate, mm / d; SGR is the specific growth rate, %; L t L0 represents the average body length of artichokes in each group on day 7 (mm); L0 represents the average body length of artichokes in each group on day 1 (mm); and t represents time (d).

[0124] As shown in Tables 1 and 4, lactic acid can improve the average daily growth rate and specific growth rate of Artemia spp. Among them, lactic acid with chitosan as a carrier has the best promoting effect on Artemia spp., with the average daily growth rate and specific growth rate reaching 0.48 mm / d and 26.06%, respectively.

[0125] Table 4. Effects of lactic acid-carrier on growth parameters of Artemia esculenta.

[0126]

[0127] Using Example 2 (LS group) as an example, the inventors further studied the synchronicity of brine shrimp hatching and development in conjunction with Comparative Example 1 (blank control group). Images were taken 24 hours after the brine shrimp eggs hatched. See details below. Figure 6 .like Figure 6 As shown, in the comparative example 1 (blank control group), some Artemia eggs remained in the umbrella-shaped stage after 24 hours of incubation and did not fully hatch; while in the LS group, the Artemia eggs fully hatched and were in the active nauplius stage 1. This indicates that lactic acid can accelerate the hatching and development of Artemia.

[0128] In summary, lactic acid can increase the hatching and development speed, average daily growth rate, and specific growth rate of artichokes.

[0129] Example 10: Transcriptome sequencing analysis and qRT-PCR verification of cultured Artemia worms from Examples 1-6 and Comparative Examples 1-2

[0130] According to Examples 1-6 and 9, using lactic acid-chitosan or lactic acid-SiO2 as antibiotic alternatives for Artemia culture significantly improved the body length and growth of Artemia, achieving unexpected technical results. To investigate the underlying mechanisms, the inventors performed transcriptome sequencing analysis on the Artemia cultured in Examples 1-6. The transcriptome sequencing results, for the first time, elucidated the molecular mechanism by which lactic acid regulates Artemia growth and immune protection at the gene level, revealing the physiological response characteristics of Artemia to lactic acid.

[0131] The following detailed explanation uses Example 2 (LS group) and Example 5 (LC group) as examples, combined with the blank control group (without the addition of the alternative anti-antibody product):

[0132] (1) Transcriptome sequencing analysis of cultured Artemia worms in Examples 1-6 and Comparative Examples 1-2

[0133] Starting from the hatching of the artichokes, sampling was conducted on day 7 for each experimental group. 300 artichokes were randomly selected from three replicates in each group, mixed, repeatedly rinsed with ultrapure water, placed in 1.5 mL EP tubes, flash-frozen in liquid nitrogen for 30 minutes, and then stored at -80 ℃ for later use. After all sampling was completed, Shanghai Meiji Biotechnology Co., Ltd. performed artichoke transcriptome analysis. 1) Functional annotation statistics: The obtained transcripts were compared in six databases, including NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG; 2) Expression analysis: Based on the quantitative expression results, differential gene analysis was performed between groups to identify differentially expressed genes. Differential analysis was conducted using DEGseq software; further KEGG enrichment analysis was performed on the differentially expressed genes. (See details...) Figure 7-9 .

[0134] Appendix Figure 7 This is a KEGG enrichment analysis diagram of differentially expressed genes in Artemia salina between the LS group and the control group. Figure 7 As shown, in the comparison between the LS group and the blank control group, differentially expressed genes were annotated into 138 pathways. Among the top 30 pathways with high enrichment, 25 were signaling pathways related to growth and metabolism. The top three pathways in terms of enrichment were the glucagon signaling pathway, fatty acid degradation, and adipokines signaling pathway. Five immune-related signaling pathways were identified, with the top three in terms of enrichment being the PPAR signaling pathway, AMPK signaling pathway, and C-type lectin receptor signaling pathway. Specifically, the LS group showed significant upregulation of CPT1A and ACACA genes in the glucagon signaling pathway, fatty acid degradation, and adipokines signaling pathway (P < 0.05). The significant upregulation of CPT1A and ACACA genes indicates dual activity in fatty acid synthesis and degradation, enabling artichokes to rapidly obtain energy in a short time and maintain a long-term energy supply through fatty acid synthesis, thus helping artichokes maintain growth and reproduction and improving their survival rate. This dynamic balance of lipid metabolism plays a crucial role in the rapid growth and maintenance of high energy requirements during the hatching period of artichokes.

[0135] Appendix Figure 8 This is a KEGG enrichment analysis diagram of differentially expressed genes in Artemia salina between the LC group and the control group. Figure 8As shown, in the comparison between the LC group and the control group, differentially expressed genes were annotated into 339 pathways. Among the top 30 pathways with high enrichment, 24 were signaling pathways related to growth and metabolism. The top three pathways in terms of enrichment were the insulin signaling pathway, the thyroid hormone signaling pathway, and carbohydrate digestion and absorption. Six were signaling pathways related to immunity. The top three pathways in terms of enrichment were the PI3K-Akt signaling pathway, the mTOR signaling pathway, and EMC receptor interaction. Specifically, in the LC group, the PI3KCA gene was significantly upregulated in the insulin signaling pathway, the thyroid hormone signaling pathway, and the carbohydrate digestion and absorption pathway (P < 0.05). The TSC2 and mTOR genes were significantly upregulated in the insulin signaling pathway and the thyroid hormone signaling pathway (P < 0.05). The MGAM gene was significantly upregulated in the carbohydrate digestion and absorption pathway (P < 0.05). Upregulation of PI3KCA, mTOR, and MGAM increases the efficiency of carbohydrate digestion and absorption, enhances insulin signaling, and further promotes glucose uptake and utilization, protein synthesis, and cell proliferation. This enables Artemia to utilize energy more efficiently, enhances energy metabolism and cell growth, and allows Artemia to develop at the fastest speed and maintain survival rate under conditions of sufficient nutrition and intensive farming.

[0136] Compared to the LS group, the LC group showed significantly higher enrichment of the KEGG pathway, a result consistent with the superior body length-promoting effect of the LC group on the experimental animals. This indicates that lactic acid, carried by chitosan, can trigger a broader biological response, activating more signaling and metabolic pathways (such as the insulin signaling pathway, carbohydrate digestion and absorption), further promoting cell proliferation, protein synthesis, and energy metabolism, thereby supporting the growth of Artemia elegans body length. In contrast, the LS group showed a lesser effect in promoting growth and development.

[0137] (2) qRT-PCR verification

[0138] To verify the reliability of the transcriptome sequencing results, one differentially expressed gene closely related to metabolic or immune pathways was selected from each experimental group for qRT-PCR verification. The specific method was as follows: Total RNA was extracted from Artemia elegans tissue using the Trizol method, and the RNA was reverse transcribed into cDNA using the PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TAKARA). qRT-PCR was performed on a LightCyeler 96 (Roche, Germany) using the TB Green® Premix Ex Taq™ II FAST qPCR (TAKARA) kit. PCR primers were designed and synthesized by Qingdao Platinum Biotechnology Co., Ltd., with β-actin as the internal reference gene. Primer sequences are shown in Table 5. Relative quantification methods (2...) were used... -ΔΔCT The fold change of the target gene was determined. T-test analysis was performed using SPSS 26.0 software; p < 0.05 was considered statistically significant. See details for the results. Figure 9 .

[0139] Table 5. Primer sequences used for qRT-PCR

[0140]

[0141] Appendix Figure 9 The results of qRT-PCR for differentially expressed genes in Artemia salina are shown. Compared with the blank control group, the CPT1A gene was significantly upregulated in the LS group (P < 0.05). Figure 9 A); PI3KCA gene was significantly upregulated in the LC group (P < 0.05) Figure 9 B). This indicates that the PCR validation results are consistent with the differential gene expression upregulation results of the transcriptome sequencing analysis, confirming the reliability of the transcriptome sequencing analysis results in this application.

[0142] In summary, this application first provides the application of lactic acid in brine shrimp farming. This application achieves both the antibacterial effect of lactic acid and stable water pH without the addition of acid-base regulators, overcoming the technical problems in existing technologies where increasing lactic acid concentration leads to a sudden drop in water pH, poses safety risks to brine shrimp, and insufficient antibacterial effect when concentration is reduced. Based on this, this application provides a method for farming brine shrimp using lactic acid as an antibiotic alternative. This farming method, by regulating the expression of growth, immunity, and other related genes, not only significantly improves the hatching and survival rates of brine shrimp and effectively reduces the number of Vibrio bacteria carried by brine shrimp, but also promotes the synchronicity of brine shrimp growth and development, enhances their immunity, and ensures the healthy and sustainable farming of brine shrimp. Compared with existing technologies that only utilize the antibacterial properties of organic acids, this farming method achieves unpredictable technical effects, providing a theoretical basis for further optimizing brine shrimp farming technology.

Claims

1. Use of lactic acid in the breeding of Artemia, characterized in that: The lactic acid is directly added into the aquaculture water with silica or chitosan as the carrier; when the lactic acid is carried by silica, the concentration of the lactic acid in the water is 180-300 μM; the weight-volume ratio of SiO2 to lactic acid is (0.5-3) g:1 ml, and the particle size of the silica is 10-40 μm; when the lactic acid is carried by chitosan, the concentration of the lactic acid in the water is 230-600 μM, the weight-volume ratio of chitosan to lactic acid is (0.01-0.1) g:1 ml, and the degree of deacetylation of the chitosan is 75-88%, and the molecular weight is 100-180 kDa.

2. Use according to claim 1, characterized in that: The density of the artemia eggs in the aquaculture water is not more than 1000 eggs / mL.

3. A simple, healthy method of breeding brine shrimp, characterized by: The method comprises the following steps: (1) mixing the carrier SiO2 or chitosan with lactic acid according to a certain weight-volume ratio to obtain a lactic acid product in the form of emulsion or powder; when the lactic acid is carried by silica, the concentration of the lactic acid in the water is 180-300 μM; the weight-volume ratio of SiO2 to lactic acid is (0.5-3) g:1 ml, and the particle size of the silica is 10-40 μm; when the lactic acid is carried by chitosan, the concentration of the lactic acid in the water is 230-600 μM, the weight-volume ratio of chitosan to lactic acid is (0.01-0.1) g:1 ml, and the degree of deacetylation of the chitosan is 75-88%, and the molecular weight is 100-180 kDa; (2) adding the lactic acid product of step (1) into the water for artemia hatching and aquaculture to obtain a lactic acid aquaculture water, wherein the concentration of the lactic acid in the aquaculture water is as described in claim 1; and (3) putting artemia eggs into the lactic acid aquaculture water of step (2) to hatch at 28-30°C for 18-24 h, and feeding the artemia with basic feed under continuous light conditions to obtain artemia.

4. The method of breeding Artemia according to claim 3, characterized in that: The density of the artemia eggs in the water in step (3) is not more than 1000 eggs / mL.

5. The method of breeding Artemia according to claim 3, characterized in that: The continuous light condition in step (3) is 500-2500 Lux; and the basic feed is Chlorella, yeast powder, spirulina powder, rice bran, soybean meal, or artificial micro-particle feed.

6. The use of Artemia in fish and shrimp farming, characterized in that: The artemia is obtained by the aquaculture method described in any one of claims 3-5.

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