The usage of probiotic microorganisms in production technology of European grayling fish stock

European grayling is a perspective object of a recreational fishing. In addition, the presence of this species in sufficient quantities in rivers has a positive influence on the formation of the recreational attractiveness of mountain regions, where tourism is one of the main sources of local communities existence. However, grayling stocks in the watercourses of Ukraine are significantly depleted nowadays, and populations are in a condition of lack, what has led to the inclusion of this species into the Red Book of Ukraine. Working out of biotechnologies of artificial reproduction of rare fish species in aquaculture conditions for the further reintroduction of the resulting offspring into natural water bodies takes an important place in the system of environmental protection measures to preserve biodiversity. After all, the reintroduction into nature of artificially obtained fish fry is one of the effective ways to restore the optimal number and structure of rare fish species natural populations, and this, in turn, will allow in the future to restore the limited exploitation of the stock of these fish species. One of the critical artificial fish reproduction technology links is the transfer of larvae to external nutrition. Namely on this stage the highest mortality rates are observed. The usage of live feeds as a starter fodder can significantly improve the situation. Forage organisms, in addition to having high nutritional value, can serve as delivering tools of various targeted products, including probiotics, to a fish larvae organism. The assessment of the usage of probiotic strain Lactobacillus casei IMV B-7280 feasibility is carried, and also the possibility of usage the culture Daphnia magna Straus, 1820 grown in the presence of trehalose lipids, within the technology of early feeding of the European grayling Thymallus thymallus (Linnaeus, 1758). It was shown that the larvae of the European grayling, which received investigated probiotic microorganisms strain, had 15% bigger weight on the final stages of feeding than the larvae of the control group. Introduction to the diet of grown-up grayling larvae of the culture Daphnia magna, previously received of common cultivation with microalgae Desmodesmus armatus (Chodat) E.H.Hegewald 2000 with addition of trehalose lipids, contributed to the reduction of larval mortality and the acceleration of their growth rate. Further improvement of proposed approaches of growing grayling fish planting material can be extrapolated to other fish species and in the future may be applicable in the process of growing rare fish species in conditions of farm aquaculture fish farms, what will allow them to be involved into environmental protection activities by placing appropriate orders. This, in its turn, will contribute to the stable development of communities in rural areas.


Introduction
The development of methods and practical measures for gene pool preservation and population size restoration of rare and vulnerable aboriginal fish species requires the implementation of integrated research, among which the work out of reproduction biotechnologies of such species in the conditions of aquaculture, takes prominent place.European grayling, in addition to having high food and taste qualities, is a promising object of recreational fishing.Due to this fact, the presence of this species in sufficient quantity in rivers has a positive influence on the formation of recreational attractiveness of mountain regions, where one of the main sources of existence of local communities is tourism.However, nowadays grayling stocks in the watercourses of Ukraine are currently significantly depleted, and populations are in a condition of lack, what has led to the inclusion of this species into the Red Book of Ukraine.Restoring the optimal number and structure of natural populations is one of the main prerequisites for removing the species from the protection lists, including from the Red Book of Ukraine, and will allow limited exploitation of fish stocks in the future.
Usage of aquacultural methods of supporting the quantity of rare and endangered fish has found an application in the countries of the European Union.Thus, due to the artificial fish reproduction and the growing of resulting offspring to a viable condition for further stocking in the Baltic countries, it became possible not only to restore the normal population size of brown trout, and also to implement licensed fishing for amateur fishermen.There is the same situation with the population of Danube salmon in Slovakia.
The development of correction technologies of live feeds nutrient composition in order to improve their nutritional value will allow to increase the young fish survival on early stages of development and thereby to reduce the cost of receiving fish planting material works.Improved and developed technologies and approaches of receiving and growing rare fish species planting material to a viable condition, including the European grayling, in future will find an application in the process of growing these species in conditions of aquaculture fish farms, what will allow in the future find application in the process of growing these species in the conditions of farm aquaculture fish farms, which will allow them to be involved into environmental protection activities by placing appropriate orders.This, in its turn, will contribute to the stable development of communities in rural areas.
Usage of probiotics in aquaculture as treatment-and-prophylactic means of dysbiosis correction is an efficient method of compensation of negative effect of strenuous conditions of fish keeping within industrial aquaculture.In addition to increasing various fish diseases resistance, probiotics are able to synthesize a number of extracellular enzymes, what increases the bioavailability of forages and ensures decreasing in its costs, what contributes to the intensification of fish growing processes [1].Usage of probiotics also positively influences on stabilization of digestive tract functions and improving its enzymatic activities, normalizing of mucus formation on the fish body surface and improving of the gill apparatus [2].Probiotic microorganisms contribute to enhancing of phagocytic, lysozyme, complement acrivities, and also expression of various cytokines in fishes [3].
Among the great variety of probiotic microorganisms, representatives of the genus Lactobacillus cause a substantial interest for aquaculture.It is based on the fact that there were not found any species of Lactobacillus, which could be pathogenic or conditionally pathogenic to aquatic organisms [4].Due to a number of adaptive capabilities, namely a high level of adhesion, synthesis of organic acids and hydrogen peroxide, resistance to adverse conditions of digestive tract, they are able to colonize digestive tract of hydrobionts successfully and resist pathogenic microflora [5].
One of the main technological problems of the use of probiotic preparations is related to ensuring targeted delivery of the appropriate bacteria to fish organism.Probiotics introduction directly into water leads to its dispersion in a significant volume of the environment, its 1254 (2023) 012093 IOP Publishing doi:10.1088/1755-1315/1254/1/0120933 introduction into granular forages is also problematic -in the process of extrusion, which is used in the production process of most types of modern production forages, the viability of microorganisms can significantly decrease.Live feed can serve as a reliable vector for targeted delivery of probiotics into fish organism [6].Traditionally in aquaculture brine shrimp nauplia are used as a live feed, which are of the right sizes for feeding early larvae of the vast majority of fish species [7].On the later stages of fish development, it is more expedient to use larger feed organisms, in particular various species of Cladoceran [8].It makes improving the increasing technologies for a biomass of live feeds an urgent issue, namely searching for inexpensive alternative environments, a possibility of nutrient correction, and most importantthe acceleration of cultivation rates.The usage of biosurfactants allows to improve absorption of nutrient substrates by forage organisms, in particular by daphnias, what ensures the growth rate of their biomass acceleration.Within the biotechnology of receiving biosurfactants the main method is a microbial synthesis.In particular, in such way trehalose lipids can be received.At the same time, it is important to understand whether the appropriate preparations will not have a negative impact on fish, which is fed with live feeds grown with the usage of biosurfactants.
The usage of live feeds and probiotics helps to increase the viability of young fish.This is especially relevant in production of fish planting material intended for introduction into nature.After all, the living conditions change during fish transfer from comfortable aquaculture conditions to natural water reservoirs is often the cause of large losses.Receiving of fry with high viability is especially relevant within carrying out work on a rare fish species reproduction.
Taking into account the above, the aim of the work was to assess the feasibility of probiotic strain Lactobacillus casei IMV B-7280 usage and also, the opportunity of usage the culture Daphnia magna grown in the presence of trehalose lipids, within the technology of early feeding of the European grayling Thymallus thymallus (Linnaeus, 1758).

Materials and methods
Experimental growing of European grayling larvae took place in 2021 on the State Enterprise "Trout hatchery "Lopushno" basis, which belongs to the department of the State agency of land reclamation and fisheries of Ukraine.Incubation of caviar and growing of fish planting material was carried out according to generally accepted recommendations for grayling [9].Larvae were reared in 20×40 cm trays with a water volume of 18 liters.During all the time of experimental growing the quality of water was controlled with Combo Water Testing Meter AZ-86031.
In both control and experimental groups, the initial number of larvae in trays was 500 individuals.Experimental feeding of larvae was started on the ninth day after hatching from eggs.Introduction of forage was carried out every hour from 8:00 a.m. to 7:00 p.m., alternating with starter dry forage Biomar and live feeds.As starter live feeds nauplia artemia was used, namely Artemia spp.
On the first stage of experiment with a duration of 13 days grayling larvae received corresponding dry forage Biomar and intact nauplia artemia, and larvae of experimental groupdry forage and nauplia artemia enriched by probiotic microorganisms culture Lactobacillus casei IMV B-7280.
The cultivation of artemia was carried out with common aquacultural method [10] .After the procedure of cleaning cyst sash and cysts which did not hatch, nauplia artemia were transferred to a fresh environment with the appropriate salinity, where lyophilized cultures of probiotic microorganisms with a concentration of 5×10 11 CFU/l were introduced.The bioencapsulation procedure lasted for 12 hours with constant aeration and lighting.
On the second stage of experiment with a duration of 7 days was investigated the expediency of live feeds usage within the technology of feeding grown grayling larvae.From individuals, which previously got the standard ration without probiotics, the control group of grayling larvae was created and they were fed with the dry forage Biomar, and also research group in which fish got live feeds besides the dry forage, namely Daphnia magna Straus, 1820.Feeding was carried out every hour and half during daylight hours.The initial number of grayling larvae is 200 individuals in each tray.In order to increase the nutritional value of forage organisms, daphnia was cultivated together with the green microalgae Desmodesmus armatus (Chodat) E.H.Hegewald 2000 [11].Acceleration of the growth rate of biomass was provided by the addition of trehalose lipids preparation, which was received from the culture fluid of Rhodococcus erythropolis.
All researches were performed in triplicate.Effects of dietary treatment were analyzed by a one-way analysis of variance (ANOVA), followed by Tukey's or Student's post hoc test to determine significant differences.Previous to statistical analysis, data were transformed with natural logarithm if identified as non-homogenous (Levene's test) to meet the assumptions for statistical methods.Mean values were considered significantly different at p ≤ 0.05.Statistical analysis was computed using MS Excel software and STATISTICA 6.0 application package.

Ethical considerations
The research conducted in this study adhered to a comprehensive set of ethical principles and guidelines in compliance with both national regulations in Ukraine and institutional policies.Below are the specific ethical considerations and procedures that were followed throughout the experiment:

National and institutional compliance
1.All aspects of this research were conducted in strict accordance with the principles outlined in the European Convention on the Protection of Vertebrate Animals Used for Research and Other Scientific Purposes.This convention outlines the ethical treatment of animals in scientific research and serves as a fundamental guideline for our ethical approach.2. We also adhered to the relevant national legislation, specifically the Law of Ukraine "On the Protection of Animals from Cruelty".This law governs the ethical treatment and welfare of animals used in research within the jurisdiction of Ukraine.

Animal welfare and care
1. Throughout the entire duration of the experiment, we maintained a rigorous standard of constant care for the animals involved.This included regular monitoring of their health and well-being, with immediate action taken to address any signs of distress or discomfort.2. Special attention was given to ensuring that the animals did not experience any unnecessary pain, suffering, or anxiety.Procedures and handling techniques were designed to minimize stress and discomfort.3.In cases where it was necessary to euthanize animals, we employed humane methods that were consistent with internationally accepted practices to minimize suffering.4. To minimize the impact on the animal population, we carefully selected the smallest necessary number of animals required to achieve the research objectives effectively.

Personnel qualifications
All individuals involved in conducting the experiments and caring for the animals possessed appropriate educational and professional training.This ensured that the highest standards of animal welfare and ethical conduct were upheld throughout the study.

Results and discussion
At the the Trout hatchery "Lopushno" the experience of artificial reproduction of aboriginal salmon fish is accumulated, including European grayling what is highlighted in a number of scientific publications [12,13].Further researches are aimed at improving proven technologies.Special attention is paid to the problem of young fish mortality reducing and accelerating its growth rates.On this farm, the incubation of grayling caviar is carried out in horizontal tray apparatuses for salmon fish.Due to the lack of an opportunity to heat the water, the duration of incubation of eggs and hatching periods at the enterprise depend on weather conditions and may significantly differ in different years.An average embryogenesis of grayling lasts 22-24 days [13].In 2021 the interval between the beginning and the end of embryos hatching from caviar took 11 days.Larvae transition to exogenous nutrition began on the sixth day after hatching, and after 9 days 100% of survived larvae switched to external nutrition, what correspond with the data of other authors [13].Accordingly, that is why namely on the 9th day after the embryos hatching completion, experimental grayling larvae feeding was started, and was carried out in two stages.
During the experiment, the water temperature in the trays increased by approximately 2 °C, while the concentration of oxygen dissolved in the water, changed insignificantly (figure 1).In general, the quality of water corresponded to the norm [9].Research results showed that grayling larvae, which received encapsulated into artemia lactobacilli, were characterized by a higher growth rate at all stages.
Thus, with the same average initial body weight of 0.02 g, after thirteen days, individuals of the research group were 15% heavier than larvae from the control group (figure 2).It is known that probiotic microorganisms are able to product extracellular digestive enzymes, including protease [2].As a result, nutrients are engulfed and absorbed more efficiently during a simultaneous use with probiotics, and forages, enriched with them, have a higher nutritional value.
Besides, it is known that L. casei has the ability to produce a specific bacteriocin-caseicin, which has an expressive bacteriostatic and weak bactericidal action.Caseicin can be producted extracellularly as well as inside a cell in a ratio of 50 : 1 [14].It can be assumed that this bacteriocin contributes to the increased elimination of pathogenic and conditionally pathogenic microorganisms in nauplia artemia, which are received from cysts collected of natural conditions.It allows to protect young fish to some extent from pathogens which may be transmitted with The positive effect of using live feeds lies in the fact that they have the necessary set of nutrients for fish, as well as contain their own hydrolytic enzymes which improve the functional activity of fish larvae digestive tract, increase the level of feeds conversion and their digestibility.All of this contributes to the growth intensification processes of young fish.
The introduction of D. magna to ration of European grayling youth contributed to the faster accumulation of fish body weight.Thus, the average daily gain of individuals of the control group amounted 3.1 g, and the larvae of the research group -4.3 g, what is 36% more (figure 3).
Producers of a large quantity of essential micronutrients for fish are microalgae [15,16].However, due to the existing cell wall, algae are often poorly digested by fish.In return, phytomass is partially digested in a digestive tract of zooplankton, what makes it bioavailable to a fish organism [11].According to this, combined cultivation of microalgae with feed zooplankton allows to receive live feeds with improved nutritional value.
According to this, one of the problems of receiving a sufficient amount of live feeds for young fish feeding is the rapid increase of its biomass.This can be achieved with the help of biosurfactants usage.Among the wide variety of biological surface-active substances, trehalose lipids are perspective for use -it is the one of glycolipids groups, in which the carbohydrate component is represented by trehalose.Trehalose is a disaccharide in which 2 residues of D-glucose are connected by an α, α-glycosidic bond.Trehalose dissolves well in water and alcohols.According to this, its compounds with fat acids and other lipid nature substances have amphipathic properties, i.e., they dissolve in water and as well as non-polar solvents.It allows nutrients in a digestive tract to form micelles and to transform hydrophobic compounds into a soluble condition [17].The ability to form micelles, on the one hand, ensures emulsification of fats, what speeds up their digestion, and on the other hand, facilitates the transportation through biological membranes, thereby improving an absorption in intestines.
Previous researches have pointed out that the introduction of biosurfactants speeds up the growth rate of forage zooplankton cultures and also reduces the effective concentration of biocides [18].
Trehalose lipids are synthesized in significant quantities by Rhodococcus erythropolis and are accumulated in a culture liquid.The question remains whether the rests of biosurfactants or their derivatives will have a negative effect on young fish.Previous researches have pointed out that the introduction of growing grayling larvae of live feeds biomass, grown by co-cultivation with green algae in presence of trehalose lipids, intensifies the growth rate of fish larvae mass, as well as contributes to increasing of survival rate of fish of the research group in comparison with the control group of individuals (figure 4).

Conclusions
Feeding of European grayling larvae during the period of its transition to external nutrition with nauplia artemia, enriched with lactobacilli Lactobacillus casei IMV B-7280 ensures acceleration of growth processes.Introduction of Daphnia magna culture, previously received of co-cultivation with microalgae Desmodesmus armatus with addition of trehalose lipids, into a growing grayling larvae ration, contributes to reducing the mortality of young fish and accelerating its growth rate.
The usage of lactic acid probiotic microorganisms Lactobacillus casei IMV B-7280 for incapsulation in artemia nauplia, and also the procedure of co-cultivation live feeds with microalgae in in the presence of biosurfactants, may be appropriate for young fish feeding, but the effectiveness of these procedures has to be investigated for each specific species separately.

Figure 1 .
Figure 1.Dynamics of water temperature and concentration of oxygen dissolved in water during experimental European grayling larvae feeding.

Figure 2 .
Figure 2. Dynamics of body weight accumulation of European grayling larvae during feeding with artemia, non-encapsulated and encapsulated with lactobacilli.

Figure 3 .
Figure 3. Dynamics of body weight accumulation of growing European grayling larvae under conditions of adding live feed to the diet.

Figure 4 .
Figure 4. Dynamics of European grayling larvae survival under conditions of live feeds of adding live feed to the diet.