RU2614604C1 - Method for reducing number of bacteria-opportunists in growing media for marine fish larvae and their fodder - Google Patents

Abstract

FIELD: agriculture.

SUBSTANCE: before placing the eggs into the incubator, the culture of microalgae Chlorella vulgaris is pre-inoculated. The culture of chlorella is also added into rearing containers with larvae at the endogenous feeding phase. At the stage of exogenous feeding the fish larvae with rotifers, the chlorella filtrate is added to the medium. Before laying the Artemia cysts for hatching, the chlorella culture is added to the cultivation medium.

EFFECT: optimal microbiological conditions in the artificial rearing system of fish and organisms for their feeding without the use of antibiotics.

3 cl, 1 tbl, 1 ex

Description

The invention relates to mariculture, can be used in hatcheries and is intended to improve the quality of the environment for the cultivation of feed organisms and fish.

One of the most pressing problems in marine fish growing technology is the unpredictable mass mortality of larvae in the early stages of development due to outbreaks of bacterial infections. Particularly susceptible to infections are flatfish larvae, since in the early stages of development their immune and digestive systems are poorly developed. The survival, growth and development of artificially grown flounder larvae are largely dependent on microbiological conditions during embryonic development and at the stage of feeding the larvae with live food. In this regard, this invention was tested precisely in the framework of the technological scheme for growing the Black Sea Kalkan.

A known method of cultivation of marine aquatic organisms in growth tanks, closed in a common automated system. Before water is supplied to animal pools, water is filtered and treated with ozone (Automated closed recirculating aquaculture filtration system and method US 5961831 A). This method effectively eliminates bacteria and allows you to abandon antibiotics. However, this method has several disadvantages. Firstly, in some cases, automatic water purification can cause injuries to organisms, in particular caviar. Secondly, the bactericidal effect of ozone is due to the fact that it forms oxidizing compounds with the components of sea water, which destroy the shell of bacteria. The resulting compounds can also react with the shell of the egg - the chorion, disrupting its biochemical structure and changing functional properties. Therefore, despite the high bactericidal effectiveness of ozone, its use should be strictly regulated. In addition, as our studies have shown, a single treatment of the incubation medium for eggs with ozone leads to a massive non-selective decrease in the number of bacteria, and in the absence of constant ozonation after a short lag phase, an outbreak of rapidly growing opportunistic microflora is observed in the medium. In addition, our studies have also shown that ozonated water (at a level of ozone concentration within the biological tolerance of disinfected organisms) does not sufficiently eliminate bacteria attached to the surface of organisms.

A known method of artificial breeding of the Black Sea Kalkan, including the incubation of eggs in incubators, the transfer of larvae after hatching into outgrowth pools, their provision with specially prepared live food. In this method, water purification and water exchange in incubators is carried out by using a biofilter and a closed water circulation system, and disinfection is carried out by introducing antibiotics (Pat. 847961, IPC A01K 61/00, USSR). This method, from a technical point of view, is advisable. However, the disinfection of water with the introduction of antibacterial drugs does not solve the problem of bacterial contamination, since bacteria can become resistant to the antibiotics used, the balanced microbiological community of the environment is disturbed, antibiotics accumulate in cultivated organisms and can pollute the environment. The negative aspects of the use of antibiotics lead to alternative solutions to the problem of bacterial contamination of aquaculture organisms.

The objective of the method of reducing the number of opportunistic bacteria in the environments for growing larvae of marine fish and their feeds is to create optimal microbiological conditions in the system of artificial cultivation of fish and their feed organisms by reducing the number of bacteria as in artificial media for incubating eggs and growing larvae of marine fish in the early stages of development, and in the cultivation environments of their live feed organisms (microalgae, rotifers, brine shrimp).

The problem is solved in that to disinfect the medium before placing the eggs, the microalgae culture Chlorella vulgaris is pre-inoculated into the incubator (the concentration of cells in the incubation medium is ≈2⋅10 ⁵ klml ^-1 ), and they are added to the growth tanks with larvae at the endogenous feeding stage culture Ch. vulgaris at a rate of ≈4⋅10 ⁴ kl -1ml ^-1 , then at the stage of exogenous feeding of fish larvae with rotifers, chlorella filtrate is added to the medium at a rate of 50 ml⋅l ^-1 at a concentration of initial microalgae culture ≈10 ⁷ klml ^-1 ; before laying Artemia cysts on hatching culture Ch. vulgaris is added to the culture medium of Artemia at the rate of ≈0.4010 ⁶ cells⋅ml ^-1 . The method uses a culture of Ch. vulgaris at the end of the exponential growth phase. In incubators, eggs after adding the culture of Ch. vulgaris incubated for 1 hour without duct, repeating the procedure daily after cleaning the bottom of the incubators until hatching.

The main vectors for the transmission of pathogenic bacteria to fish larvae during artificial cultivation are the surface of the eggs, the growing medium, and feed organisms (rotifers, brine shrimp). In the absence of proper caviar disinfection, bacterial infections from producers to larvae can be transmitted through it.

Colonization of the surface of the eggs with bacteria (the formation of caviar epiflora) occurs within a few hours after fertilization. The bacteria that make up the epiflora produce proteolytic enzymes that can negatively affect the development of the embryo; caviar growth prevents its breathing, slowing down development and hatching. One of the alternative methods for reducing the level of bacterial contamination (and especially reducing the titer of rapidly growing potential pathogens - opportunistic bacteria) of the growing medium is to use the antibacterial properties of unicellular microalgae. The antibacterial activity of microalgae of the genus Chlorella is known, which was discovered in the 1940s (Pratt et al., 1944), however, to date, the functional mechanisms of this activity are unclear. The associated microflora of nonaxenic cultures of microorganisms of the genus Chlorella is characterized by the presence of predominantly gram-positive bacterial isolates, about 30% of which inhibit the growth of known pathogens in vitro (Makridis et al., 2006). However, axenic cultures of species of the genus Chlorella, in the absence of cultivated associated bacteria, have significant in vitro inhibition of known pathogens of fish of the genus Vibrio (Kokou et al., 2012). The bactericidal activity of chlorella, apparently, is also associated with its exometabolites, which contain up to 25-40% of polysaccharides (Lewin, 1962), a significant amount of glycolic acid (Watt, Fogg, 1966; Maximova and Dahl, 1975), as well as formic and acetic acids (Maksimova, Pimenova, 1969).

The transition of larvae to exogenous nutrition with live food is the most problematic stage of fish cultivation in relation to possible outbreaks of bacterial infections. The beginning of feeding (the opening of the digestive canal) initiates the colonization of the intestines of the larvae by the microflora associated with the growing medium and live food, which may contain conditionally pathogenic bacteria (including those transmitted through eggs from manufacturers). Under intensive growing conditions, at high densities of cultivated organisms (for example, rotifers), an environment favorable for the rapid growth of opportunistic bacteria develops and their pathogenicity increases, which leads to outbreaks of infectious diseases and mortality of larvae. A significant number of bacteria that are not always eliminated with disinfectants are also localized in the surface layers of the outer shell of the brine shrimp, a living feed organism that feeds the farmed Kalkan larvae.

The technical result manifested in the implementation of the method is that:

- application of Ch. vulgaris allows you to create optimal conditions for the cultivation of larvae by introducing its metabolites with antibacterial action into the growth medium along with the chlorella culture, as well as populating the growth media with chlorella-associated bacteria favorable for the life of the larvae (eubiotics) that inhibit the growth of opportunistic microflora;

- the formation of a healthy balanced microbiological community by introducing a non-aacenic culture of Ch. vulgaris with associated microflora when opening by mouth with larvae and passively swallowing water provides the primary colonization of the intestines of the larvae with healthy microflora and inhibits the growth of opportunistic bacteria in the growing medium when the larvae switch to exogenous nutrition;

- use of Ch. during incubation of Artemia vulgaris allows to increase the synchronization of hatching of Artemia from cysts, to reduce the level of bacteria in the environment of incubation of cysts and the intensity of bacterial colonization of the surface of nauplii and metanauplia Artemia salina - a potential vector for the transfer of pathogenic bacteria to fish larvae;

- view of Ch. vulgaris is technologically advanced, and obtaining the necessary biomass of this type of microalgae is possible in a short time;

- the conditions for the incubation of eggs, the cultivation of larvae and their food organisms are close to natural, which avoids the risks associated with the use of antibiotics or other disinfectants, and improve the quality of aquaculture products;

- decreases the intensity of bacterial colonization of the surface of the eggs and the environment of its incubation, and thereby reduces the likelihood of an outbreak of infectious diseases of fish larvae.

The inventive method meets the criteria of novelty and technical level, because for the first time, an alternative to chemical disinfection methods has been proposed for reducing the number of opportunistic bacteria at the stages of cultivation of marine fish and their food organisms that are problematic with respect to a possible outbreak of pathogenic infections; For the first time, a method for using the microalgae filtrate Ch. vulgaris as an antibacterial agent in the growing environment when feeding larvae with live feed organisms. The use of fresh filtrate allows you to reduce the level of bacteria in the environment, while maintaining the necessary balance of the number of organisms (larvae / food objects) that corresponds to the growing technology, avoiding undesirable changes in the biochemical composition of rotifers, which can occur as a result of their feeding with chlorella.

The invention is implemented as follows:

1. The incubation of eggs is carried out under standard conditions. Before being placed in incubators, eggs are washed with ozonated water to disinfect, then placed in incubators, which are previously inoculated with the microalgae culture Chlorella vulgaris, until their concentration in the incubation medium is ≈2⋅10 ⁵ klml ^-1 , and incubated for about 1 hour without duct. The procedure is repeated daily, after cleaning the bottom of the incubators until hatching. The presence of chlorella allows more than halving the intensity of bacterial colonization of the caviar surface and balancing the bacterial community in the incubation medium, since in the presence of chlorella, slowly growing, mainly K-strategists dominate in the bacterial community.

2. After transfer of hatched larvae to growth pools, on the eve of the transition of larvae from the endogenous feeding stage to exogenous feeding, to prevent an outbreak of opportunistic bacteria and to create a balanced microbial community in the water that the larvae passively swallow and which can form the primary intestinal microflora of the larvae, growth containers add Ch. vulgaris in the calculation of ≈4⋅10 ⁴ cells⋅ml ^-1 .

3. In step exogenous supply to prevent bacterial growth in the number of medium larvae, rotifers actively feeding in rearing container was added the filtrate of chlorella culture only (without cells) (50 ml⋅l ^-1, the concentration of the original culture ≈10 ⁷ kl⋅ml ^{- 1} ) since rotifers can give an additional undesirable outbreak of numbers and change their biochemical composition as a result of feeding chlorella cells to an unfavorable outbreak for fish larvae.

4. During incubation of Artemia cysts in order to obtain nauplii, the second feed organism of fish larvae, Ch. vulgaris is added to the medium before laying the cysts for hatching at the rate of ≈0.4⋅10 ⁶ cells⋅ml ^-1 . Presence of Ch. vulgaris in an Artemia incubation medium reduces the colonization of nauplii integument by bacteria, reduces the time for nauplii to hatch from cysts, and provides a more synchronized artemia metamorphosis during the transition from the nauplial to the metanauplial stage.

In all cases (1-4), a Ch. vulgaris, which is at the end of the exponential growth phase (at a concentration of 4-10 ⁷ cells / ml ^-1 ), which is preliminarily adapted for several hours to the conditions for further use (temperature, light exposure).

The proposed digital values are the result of numerous experiments conducted by the authors, allowing to complete the task as efficiently as possible.

In addition to the operations of the known technology for the cultivation of flounder / kalkan spend:

1) Cultivation of microalgae Ch. vulgaris in accordance with standard techniques;

2) Preparation of culture filtrate Ch. Vulgaris;

3) Incubation of eggs with the addition of Ch. Vulgaris;

4) Cultivation of rotifers Brachionus plicatilis in accordance with standard methods;

5) Adding filtrate Ch. vulgaris simultaneously with rotifers in the medium of cultivation of larvae;

6) Incubation of Artemia cysts in order to obtain nauplii with the addition of Ch. vulgaris.

An example implementation of the method

The proposed method for reducing the number of opportunistic bacteria in the media for growing larvae of marine fish and their feeds was successfully tested by incubating eggs and growing larvae of the Black Sea kalkan and its main food organisms (rotifers, brine shrimp) in pilot plants. Using flow cytometry with preliminary staining of bacteria with a fluorescent DNA marker, it was confirmed that in incubation of eggs, growth of larvae at the stages of endo- and exogenous nutrition, incubation of A. salina artemia with the addition of cells and Ch. vulgaris bacterial count was significantly lower than in the control. The intensity of bacterial colonization of the caviar surface and artemia nauplii was also significantly lower than that in the control.

Microalgae cultivation Ch. vulgaris in accordance with standard techniques.

Microalgae Ch. vulgaris were obtained from the collection of living cultures of marine microalgae of the Department of Ecological Physiology of Algae FSBII IMBI (Sevastopol). The cultures were grown in a cumulative mode on a Walne medium at a temperature of 24 ± 1 ° C. Lighting was carried out around the clock using LD-40 fluorescent lamps at an intensity of 5000 lux. Culture Ch. vulgaris adapted to the conditions of further use during the day. The number of cells in the initial culture of chlorella used at different stages of cultivation was 2⋅10 ⁷ -10 ⁷ cells -1ml ^-1 .

Preparation of culture filtrate Ch. vulgaris

The filtrate was prepared from a microalgae culture of Ch. Vulgaris, which is in an exponential growth phase, using 47-mm nitrocellulose membranes (pore diameter 0.2 μm) and filter equipment Sartorius (Germany). The number of cells in the original culture of chlorella, which was prepared from a filtrate was ≈10 ⁷ kl⋅ml ^-1. Cells Ch. vulgaris, separated from the filtrate, was further used in the cultivation of rotifers.

Caviar Incubation Procedure with Ch. vulgaris.

Fertilized eggs at the blastula stage were washed with prepared seawater and placed in incubators, to which Ch. vulgaris. The density of eggs in incubators was ≈1000 ind.⋅l ^-1 , of Ch. vulgaris 0.2⋅10 ⁶ cells⋅ ^-1 .

The cultivation of rotifers B. plicatilis in accordance with standard methods

The parthenogenetic clone of rotifers B. plicatilis was cultivated in a cumulative mode under constant illumination and a temperature of 24 ± 1 ° C, using prepared sea water (filtration through cartridge filters 10, 5, 1 μm, UV treatment, pasteurization).

Procedure for adding rotifers and Ch. vulgaris in the medium of cultivation of larvae.

To optimize the biochemical composition of rotifers as a food organism for fish larvae, they were preconcentrated and saturated with the corresponding types of microalgae, then washed with running seawater and placed in containers with larvae starting exogenously, into the growth medium of which chlorella filtrate was previously added. The density of the larvae was 40 ind.⋅l ^-1 . The average density of rotifers in the growth tanks of the larvae was approximately - 5 ind. экзml ^-1 . Chlorella filtrate was added to the medium with larvae at the rate of 50 ml; the concentration of the initial culture was ≈10 ⁷ cells⋅ ^-1 .

The procedure for obtaining nauplii from Artemia cysts with the addition of Ch. vulgaris.

Pre-encapsulated Artemia cysts were placed in conical vessels containing sterile sea water and kept at 28 ° C, constant illumination of 5 thousand lux and intensive sparging. The initial concentration of Artemia cysts was 1 g⋅l ^-1 , the concentration of chlorella cells added to the incubation medium was 0.4⋅10 ⁶ cells⋅ ^-1 . Hatching nauplii were separated from the shells, after which they were concentrated and washed with sterile sea water.

Thus, the use of microalgae Ch. According to the developed scheme, vulgaris allows you to create optimal microbiological conditions in the system of artificial cultivation of fish and their food organisms. This method provides a reduction in the number of opportunistic bacteria, providing a balanced bacterial community in organisms growing environments, reducing the intensity of bacterial colonization of their surface without the use of antibiotics, and, as a result, improving the quality and survival of larvae in the early stages of development.

Claims (3)

1. A method of reducing the number of opportunistic bacteria in growing media of marine fish larvae and their feeds, including incubating eggs in incubators, transferring larvae to growing pools, providing specially prepared live feeds and disinfecting the growing environment, characterized in that for disinfection of the environment before placing eggs in an incubator previously inoculated culture microalgae Chlorella vulgaris (cell concentration in the incubation medium ≈ 2⋅10 ⁵ kl⋅ml ^-1), and in nursery container with larvae in step endogenous Ethan added Ch culture. vulgaris at a rate of ≈ 4⋅10 ⁴ cells⋅ ^-1 , then at the stage of exogenous feeding of fish larvae with rotifers, chlorella filtrate is added to the medium at a rate of 50 ml⋅ ^-1 , the concentration of the initial culture is ≈ 10 ⁷ cells⋅ ^-1 medium with larvae , and before laying Artemia cysts on hatching Ch. vulgaris is added to the culture medium of Artemia at the rate of ≈ 0.4≈10 ⁶ cells⋅ml ^-1 . 2. The method according to p. 1, characterized in that the culture of Ch. vulgaris at the end of the exponential growth phase. 3. The method according to p. 1, characterized in that in the incubators of the eggs after adding the culture of Ch. vulgaris incubated for 1 hour without duct, repeating the procedure daily after cleaning the bottom of the incubators until hatching.