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View of Influence of Lactobacillus Brevis 2K Gv Lactic Acid Bacteria Strain as Part of Biomar Feed on Physiological Condition of Juvenile Parasalmo Mykiss Trout

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Influence of Lactobacillus Brevis 2K Gv Lactic Acid Bacteria Strain as Part of Biomar Feed on Physiological Condition of Juvenile Parasalmo Mykiss Trout

SIDOROVA, Natalya Anatolyevna

1*

, KUCHKO, Tamara Yuryevna

2

, MATROSOVA, Svetlana Vladimirovna

3

, VASILIEVA, Alina Valeryevna

4

1*Petrozavodsk State University, Institute of Biology, Ecology, and Agricultural Technologies, Department of Zoology and Ecology, Russia. E-mail: [email protected]

2Petrozavodsk State University, Institute of Biology, Ecology, and Agricultural Technologies, Department of Zoology and Ecology, Russia.

3Petrozavodsk State University, Institute of Biology, Ecology, and Agricultural Technologies, Research and Development Center for Aquiculture, Russia.

4Petrozavodsk State University, Institute of Biology, Ecology, and Agricultural Technologies, Russia.

Abstract

This article is a study of the influence of the Lactobacillus Brevis 2k. Gv Lactic Acid Bacteria (LAB) Strain as Part of the Biomar Feed on the Physiological Condition of Parasalmo Mykiss trout (0+). The supplement to the basic diet was administered by ingestion for 30 days by ingestion at a concentration of 2×108 L. Brevis cells per mg of feed. The result was evaluated against the feed consumption efficiency, hematologic parameters of the bloodstream, and the phagocytic activity.

A high LAB content of 29.7% in the intestine of the trout placed on the diet with L. brevis was observed for the entire test period, whereas the average LAB content in the intestine of the reference group fish placed on the additive-free diet did not exceed 2.4%. The occurrence of LAB affected an increase in the ESR and the share of leucocytes actively involved in the phagocytic reactions. The elucidated properties of Lactobacillus brevis: 2k.Gv are the strain features of the species and can be used to develop the probiotic preparation on its basis.

KEYWORDS

Biomar Feed, Lactic Acid Bacteria (LAB), Gut Flora, Physiological Parameters, Rainbow Trout.

Introduction

Nowadays, the optimization of commercial trout breeding has to do not only with the increased size of production operations but with preventing epizootic risks and the resistance of pathogens to antibacterial preparations, the impairment in the trout’s tolerance to environmental stresses, and the negative changes in the environmental state of water reservoirs used for fish breeding [Sоrum, 2006]. At the same time, it is undisputably proven by the results of fish immune status tests that the breeding conditions must favor the activity of the immune system by positively affecting both, the cellular and humoral factors of constitutive and specific immunity [Vikhman, 1984; Mikryakov, 1984; Strelkov, 1992; Sakai, 1999; Kitashova, 2002; Burgos et al., 2004]. This is due to the fact that the protective and adaptive mechanisms of fish largely depend on external effects, whereas the set of abiotic and biotic environmental factors is an active immune activity regulator. In special cases, when the breeding technology is violated, the diet changed, the quality of the habitat transformed, or inefficient antibiotics treatment procedures are applied using chloromycetin, biomycin, oxytetracycline, and compound feed with feed grisin or biomycine, the fish is stated to suffer from immunosuppressed conditions, cumulative accretion of toxic compounds or residual fractions of antibacterial preparations in tissues, which eventually impairs the consumer appeal of fish products [Navashin, 1988; Misra et al., 2006].

To improve immunoreactivity, overall tolerance, and quality of measures taken to prevent stress-related diseases among aquiculture items and reduce negative risks of commercial fish breeding, several guidelines have been elaborated for placing the fish on a curative and preventive diet with probiotics as part of commercial products based on such bacteria, as Azomonas sp., Bacillus sp., Lactobacillus sp., Enterococcus sp., Carnobacterium sp., Saccharomyces cerevisiae yeast fungi, and other species of microorganisms [Cruz et al., 2012; Barulin et al., 2016].

For some examples of commercial preparations with probiotics tested, certified, and recommended for use in fish breeding see Table 1.

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Table 1. Preparations with probiotics for curative and preventive fish feeding

No. Preparation Microorganisms Preparation purpose Source 1 Azogilin Azomonas agilis и

Lactobacterium acidophilus

Prevention and treatment of infectious diseases

[Karaseva, 1994]

2 Bacell

Bacillus subtilis, Lactobacillus aci-dophilus, Ruminococcus albus, European sprat, sunflower, cereal and legume crop processing products

Optimizing physiological condition, improving hematologic and fish breeding parameters.

[Gutsulyuk, 2014]

3 Alchem Poseidon

Bacillus subtilis, Lactobacillus acidophilus, Сlostridium butyricum, Saccharomyces cerevisiae

Improving cellular and humoral factors of non-specific tolerance:

lysozyme activity, migration of neutrocytes, bactericidal activity of blood serum

[Taoka et al., 2006]

4 Bactocell Pediococcus acidilactituma Optimizing dietary structure. [Hosseini, 2019]

5 Biogen Bacillus subtilis + hydrolases Optimizing dietary structure,

improving productivity. [Haroun, 2006]

6 Biostart Bacillus sp. Optimizing cultivation. [Queiroz,1998]

7 Cernivet LBC Enterococcus faecium SF68 Bacillus toyoi

Edwardsiella tarda prevention and treatment

[Chang et al., 2002]

8 Vetom 1.1 Bacillus subtilis и Bacillus licheni-formis

Prevention and treatment of mixobacteriosis (including the one caused by Flavobacterium

psychrophilum), post-stress and post- toxipathy recovery of physiological condition.

[Nechaeva, 2014]

9 Lactobacterine

Lactobacillus plantarum 8Р-АЗ Lactobacillus fermentum 90Т- С4

Bacterial vegetation recovery, positive effect on the body, improved overall tolerance.

[Grishchenko, 1999]

10 Olin

Bacillus subtilis (RNCIM 10172) and Bacillus

licheniformis (RNCIM 10135)

Prevention and treatment of infectious diseases, enrichment of digestive system with such enzymes, as amilase, lipase, protease, pectinase; refilling the body with essential amino acids (alanine, valine, tyrosine, treanine) and group В vitamins; recovering normal gut flora, improving immune status.

[Minkh, 2019]

11 Prolam

Lactobacillus delbrueckii subsp.

bulgaricus, Lactobacillus acidophilus, Lactococcus lactis subsp. lactis, Bifidobacterium animalis

Optimizing physiological condition, improving hematologic and fish breeding parameters.

[Gutsulyuk, 2015]

12 Subalin (vetosubalin)

Bacillus subtilis VPKM V-4759 (cryptogamous)

Prevention of skin and gill apparatus diseases, stabilization of the intestine function, recovery of the natural balance between the normal and the potentially pathogenic gut flora.

[Patent 1839459]

13 STF-1/56 Enterococcus faecium

Prevention of contagion with salmonellosis and copiseoticaemia originators, prevention and treatment of dysbacteriosis; recovery of metabolic function in the intestine, digestion, vitamin absorption.

[Patent 2698189]

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In addition, the polycomponent probiotic additives based on such bacteria, as Bacillus sp., Pediococcus sp., Enterococcus sp., Lactobacillus sp., are known to affect the morphophysiological parameters of juvenile trout [Ozorio et al., 2015]. For the published results of the studies of the effect of probiotics on the fertility function, growth and biochemical parameters of P. mykiss trout and their antagonistic activity against pathogenic bacteria and some viruses see [Gatesoupe, 1999; Verschuere et al., 2000; Irianto et al., 2002; Vine et al. 2006].

A lot of probiotic preparations used in fish breeding contain LAB (Table 1). As a rule, lactic acid bacteria with probiotic potential are isolated from natural microbial communities with the help of complex media with relatively large amounts of yeastnel, milk whey, or blood. The distinct features of LAB are lactate formation during fermentation, exigence to growth factors (lactoflavin, thiamine, pantothenic, nicotine, and folic acid, etc.); absence of cytochrome-type hemoproteins and catalases and endospores (except for Sporolactobaсillus inulinus) [Komkova, 2017]. According to multiyear study results, the positive effect of LAB on the organism is related to the biosynthesis of antibiotic-like compounds and also to the production of a large range of organic acids and change in the pH value, formation of Н2О2, reduction in the oxidative-reductive potential of the medium, and struggle for adhesion sites and nutrients [Fuller, 1989; Tarakanov, 2007].

The positive effect of LAB on the physiological status of aquiculture items and tolerance to unfavorable environmental factors, infectious diseases included, has been taken notice of in many papers [Robertson et al., 2000;

Nikoskelainen et al., 2003]. According to the findings obtained by [Nikoskelainen et al., 2001], the addition of L.

rhamnosus in an amount of 109 and 1012 CFU per mg of feed for 51 days to the diet of rainbow trout reduces the mortality caused by A. salmonicida from 52.6% in the control group to 18.9 and 46.3% in the test groups, respectively. It was also found out that the LAB specimens from such genuses, as Lactococcus, Leuconostoc, Lactobacillus, isolated from the intestine of salmonids show a strong level of adhesion to GIT cells, behave antagonistically with pathogens, and are highly tolerant to рН of 3 and 10% in-vitro fish bile [Balcázar et al., 2006].

As found out by [Arijo et al., 2008; Brunt et al., 2008; Merrifield et al., 2011], the presence of LAB stimulates the leading immunological and hematologic processes in the fish organism.

This paper exposes a series of aquarian tests and the primary findings of the effect of Lactobacillus brevis: 2k.Gv as part of the extruded Biomar feed on the growth and evolution of juvenile Parasalmo mykiss trout and also on some morphological and immunological parameters of fish blood for expanding the range of curative and preventive feed supplements based on probiotic germ cultures. To assess for efficiency the inoculation ofLactobacillus brevis to the feed, several tasks were set forth, namely:

1. compare by analysis the efficiency, with which the standard BioMar feed was consumed by the control group of juvenile trout, to the efficiency, with which the basic feed enriched with Lactobacillus brevis was consumed by the test group;

2. evaluate the changes in the weight parameters and physiological indicators of the feed consumption by juvenile rainbow trout upon the addition of Lactobacillus brevis to the basic diet;

3. describe potential clinical and ichthyo-pathological changes in the organism of the trout placed on the curative and preventive diet;

4. analyze the effect of the change in the diet on the cellular composition and hematologic parameters of the bloodstream in Parasalmo mykiss;

5. evaluate the changes in the activity of the cellular factors of inherent immunity in case of using the compound feed with Lactobacillus brevis;

6. examine the change in the biodiversity of the trout intestine flora upon adding Lactobacillus brevis to the diet.

Materials and Methods

The influence of L. brevis on the physiological condition of juvenile P. mykiss was studied at the Research and Development Center (RDC) for Aquiculture of the Petrozavodsk State University. The strain used for the test was L.

brevis: 2k.Gv, isolated from the enrichment lactic fermentation culture at the RDC’s micriobiolgical laboratory and then identified in the molecular diagnostics laboratory of the Core Facility “Bioengineering” of the Federal Research Center “Fundamentals of Biotechnology” (Moscow). The genotyping assay was conducted using the technique elaborated by [Sanger F. Et al., 1977] and with the help of the BigDyeTerminatorv.3.1 reagents kit

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(AppliedBiosystems, Inc., USA) on a ABIPRIZM 3730 genetic analyzer (made by AppliedBiosystems, Inc., USA) according to the manufacturer’s guidelines.

L. brevis: 2k.Gv was found out to show frank enzymatic activity and be capable of selectively suppressing the growth of such indicative microorganisms, as Escherichia coli, Staphylococcus aureus, Bacillus subtilis, which allows considering the strain in question as a potential probiotic for inclusion into curative and preventive supplements to feeds for aquiculture, trout included. In the trout feeding test the stationary conditions of cultivation for accumulating the necessary biomass of L. Brevis were formed in a RTS-1C (BIOSAN) bioreactor with software and the online control of microbiological growth control. The LAB were incubated using sterile 50 ml TPP TUBESPIN 50 tubes with a membrane filter (TubeSpin Bioreactor 50, TPP) for aerophilous cultivation.

When the cultural medium’s maximal optical density of 0.98–1.2 optical density units (ODU) was reached, the fermentation was stopped, the cells were concentrated for ten minutes in a SIGMA 1-15P centrifuge with microprocessor control at 3 000 rpm, and the deposit was twice washed and resuspended in the buffer solution with a pH of 7.2 and Na3PO4 and NaCl concentrated at 10 and 150 mmol/l, respectively.

The test object sample consisted of 40 juvenile 0+ trouts with an average weight of 93.96 ± 7.03 g. Before the test the fishes were randomly distributed in two representative groups (test and control) and across four 100-l aquariums with ten specimens in each. The aquaria were used to make up two independent closed-loop water supply units (CLWSU) with a system of water recirculation at 2 l/min and oxygen supply with the help of an oxigenator. The fish restricted to the diet with the LAB supplement (test group) was placed in the first CLWSU (aquaria 1 and 2); the fish restricted to the diet without the dietary supplement (control) was placed in the second CLWSU (aquaria 3 and 4).

The trout was kept at 13.2 ± 0.5С, a dissolved О2 and general NH3 concentration of 8.8 ± 0.4 and 0.01 ± 0.007 ml/l, respectively, and in natural ambient light. The alimentation of the test and the control groups fish was begun after ten days of adaptation. The control group was alimented with the extruded BioMar feed with 3 mm pellets made in Denmark. For the nutrient composition of BioMar see Table 2.

Table 2. Properties of BioMar Efico Alpha 790 feed with 3 mm pellets

No. Nutritive indices Amount

1. Crude protein, % 44-47

2. Crude fat, % 25-28

3. Crude fiber, % 1.3-4

4. Ash, % 4-7

5. Hydrocarbons (nitrogen-free extractive fraction), % 15-18

6. Phosphorus, P % 0.9

7. Digestible energy, MJ/kg 21

8. Digestible BioMar energy (total energy), MJ/kg 19.7

The test group was alimented with the BioMar feed with original L. brevis: 2k.Gv. The minimal bacteria content in a daily portion of the feed was 108 CFU/mg. The pellets and the bacteria concentrate were mixed before use and then left for a 12-hour dressing at a room temperature of 21±3°С. The fish was alimented with the BioMar extruded feed with L. brevis for 20 days.

The daily feeding rate was calculated proceeding from 1.5% of the fish body weight. The feed was administered in equal amounts in both groups. For the general alimentation test pattern see Table 3.

Table 3. Test pattern Fish

group Feed administering parameters

Control BioMar compound feed with the nutritional value in line with the recommendations for the juvenile trout breeding on the basis of the daily feed rate at a water temperature of13С.

Test

BioMar compound feed with the nutritional value in line with the recommendations for the juvenile trout breeding on the basis of the daily feed rate at a water temperature of13С, with L. brevis: 2k.Gv added to the daily feed portion in an amount of at least 2-4 × 108 CFU/mg.

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The behavior of the fish and its reaction to the feed were tracked every day of the test. To evaluate the efficiency of the feed consumption spent on growth, the feeding ratios (FR) and the fish growth rate were calculated in absolute and relative units [Kolmatskii, 2018; Pravdin, 1966] in both groups. All of the fish weight measurements complied with GOST 1368-2003.

The feeding ratio was found as FR = Pfdu, where Pfd is the administered feed weight in g and Рu is the biomass increment in g.

The absolute average daily increment in the fish body weight was found as C (g/dly) = (Mi − M0)/t, where М0 is the fish weight in the initial feeding instant in g, Мi is the fish weight in the final feeding instant in g, and t is the feeding period in days.

The relative average daily increment was found as Сw (%) = 2 (Mi − Mо) ×100, where (Mi + Mо) t

Мо s the fish weight in the initial feeding instant in g, Мi is the fish weight in the final feeding instant in g, and t is the feeding period in days.

The trout’s physiological condition was evaluated, considering the fish’s survival rate; the dormant fish was removed right away and the survival rate found as Survival rate (%) = (Ni / N0) ×100, where Ni and N0 are the amounts of fish at the beginning and end of the test, respectively.

In case of fish kill the dead specimens were exposed to a thorough clinical checkup and ichthyopathological examination. The procedures conducted for the purpose included describing the condition of the gills, the occurrence of flushing, punctate and macular extravasations on the skin and the intestine lining, the condition of the liver, kidneys, and spleen, and the inflammatory processes in the swim bladder (Fig. 1).

a b

c d

Figure 1. Shots from the clinical checkup and ichthyopathological examination: the flushing in the great vessels, the condition of the internals, the anaemia of the gills, and the structure of the liver and spleen are shown, respectively,

in shots a, b, c, and d

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The liver and spleen were obligingly weighed for measuring the absolute biological parameters and calculating the hepatosomatic and the spleen somatic index (HPSI and SPSI) as the relation of an organ’s weight to the fish weight in grams, that was expressed in percents [Handbook on Fish Physiology, 1986; Chusovitina et al., 2008]:

HPSI = Ml / M × 100, where Ml and M are the liver and the fish weight in g;

SPSI = Ml / M × 100, where Ml and M are the spleen and the fish weight in g.

In addition, we conducted the real-time monitoring of the water medium during the whole test by such parameters, as temperature mode data, transparency, dissolved oxygen content in water, pH, and concentration of ammonia nitrogen and nitrite nitrogen. The background gut flora was analyzed by taking samples from the examined trout according to aseptic regulations within 10 and 20 days and placing the samples to toss-away tubes with a carrying nutriculture medium. The flora’s taxonomic composition was evaluated according to the guidelines “Methods of Clinical Research of Opportunistic Pathogens in Clinical Microbiology” (1991) and on the basis of the phenotypical criteria specified in Bergy’s manual (1997).

The cellular composition and hematologic parameters of the bloodstream in the Parasalmo mykiss from both groups were analyzed by taking two blood samples from the heart per variant with the help of one-off 3 ml sterile syringes with a needle. The blood was stabilized using a blood thinner in the form of 3 % Na3C6H5O7 sodium citrate solution.

All the hematological tests were conducted by the standard methods used in ichthyology [Ivanova, 1982; Zhiteneva, 1989]. The ESR was measured by Panchenkov’s micromethod.

According to the recommended practices of hematologic evaluation of fishes (1999), the ESR of small trouts was measured on a smaller volume of blood (1/2 К) while maintaining the Na3C6H5O7-blood ratio of 1:2. The blood cells were identified according to the recommended blood atlases of N. T. Ivanova (1982), while the changes in the formed elements were identified according to the guidelines from L. T. Zhiteneva (1989). The Pappenheim stained blood preparations were analyzed by immersion using a Motic DM-BA-300 optical microscope with a Moticam T camera at a zoom-in of ×1000. The microscopy findings were used to identify and calculate the percentage ratio of erythrocytes, immature lymphocytes, mature lymphocytes, monocytes, granulocytes, and blood disks.

The cell factors of inherent immunity were evaluated by the in-vitro phagocytic activity control method introduced by E. A. Kosta and M. I Stenko (1947). The analyzed phagocytic activity parameters were the phagocytic index (PHIndex), phagocytic activity (PHActiv), and phagocytic intensity (PHIntens). The PHIndex was defined as the ratio of the Ssum of phagocyted cells of a phagocytic activity object to the total number of neutrophils (N):

PHIndex = S/N.

The phagocytic activity was calculated as the percentage relation of the neutrophils involved in phagocytosis to the total number of counted cells:

PHActiv = Ninv/N100%%.

The phagocytic intensity was calculated by dividing the amount of the phagocyted object by the number of neutrophils involved in phagocytosis:

PHIntens = SumSt/PHActiv = S/ Ninv.

The statistical analysis of the results was made in STATISTICA 6.0 and MS Excel: the calculated parameters were the medium values (M), the error in arithmetic average (±m); and the certainty of differences according to Student’s t-test at a probability of 95 % [Ivanter, 1992]. The biodiversity and the numerical values of the basic properties of the BG flora of the P. mykiss intestine were analyzed by calculating Simpson’s (Simpson – 1-D), Shannon’s (Shannon – H), Menhinick’s (Menhinick – Dmn), Margalef’s (Margalef – DMg) indices and also Pielou's evenness index [Magurran, 1992].

Results

For the results of the test aimed at measuring the influence of the Lactobacillus brevis added to the BioMar feed on the growth and survivability of juvenile rainbow trout see Table 4. It has been found out that, despite certain differences in the initial fish weight (90.27 and 97.64 g for the test and the control group fishes, respectively), not only did the average weight of the trouts from the test group equalize with the control values but even surpassed them by 4.72 g. In addition, the test group’s trouts achieved higher absolute and relative increments by1.97 g/day and

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2.18 %, respectively. The findings correlate with the results of analyzing the survival rate in the test and the control group. At the end of the test the survival rate in the test group was 45 %, i.e., by 60 % higher than in the control group.

Table 4. Growth and survival rates of juvenile P. mykiss trout in the test and the control group (average values) no. Parameter Trout group

Test (n = 20) Control (n = 20) 1 Initial weight, g 90.27 ± 7.61 97.64 ± 6.44 2 Final weight, g 129.73 ± 16.64 125.01 ± 15.58 3 Absolute increment, g/day 1.97 ± 0.59 1.37 ± 0.61

4 Relative increment, % 2.18 1.40

5 Survival rate, % 45 27

6 Overall increment, g 39.46 27.37

According to the analysis of the feeding ratio (FDR), both groups showed good levels of assimilating the nutrients from BioMar both, with and without the LAB supplement (Table 5). In both cases the FDR was below the basic level of 1.39 calculated for breeding trout in natural water reservoirs to a weight of 100 to 200 g at 12 ºC. That said, in the test group placed on the diet with LAB the feed conversion was by 50 % more efficient than in the control group (the lower is the FDR, the more efficient is the feed consumption), which confirms the positive effect of adding LAB to the diet of juvenile trout.

Table 5. Changes in the feeding ratio in the test and the control group No. Parameter Fish group

Test (n = 20) Control (n = 20) 1 Amount of feed, g 34.4 34.72

2 Feeding ratio 0.87 1.27

The changeability of the individual growth parameters of any organism, fish included, is known to be affected by a set of biotic and abiotic factors and plays an important part in the adaptation to the growth environment, including dietary changes [Stroganov, 1962; Shulman, 1972], and also depends on the physiological condition [Dzyubuk et al., 2015].

To study the intensity, with which the feed’s components affect the ontogenetic processes in the juvenile trout organism, we calculated such parameters of its internals as the hepatosomatic and the spleen somatic index (HPSI and SPSI). As shown by the test findings, by the end of the test both they exhibited a positive behavior in both groups (Table 6). The HPSI of the trout from the test and the control groups increased for the 20 days of the test by 23 and 37 %, respectively, which indicates that reserve nutrients accumulated in the trout liver as fat and glycogen. It is possible that the lower HPSI among the test group trouts as compared with the control group had to do with including LAB in the systemic metabolism; in its respect, this does not rule out the control by the flora over the storage of lipids in the liver.

Table 6. Average body organ indices of juvenile P. mykiss trout

Fish group HPSI, % SPSI, %

Pre-test 1.2 ± 0.08 0.09 ± 0.01

Test (at the end of the test) 1.48 ± 0.11 0.16 ± 0.02 Control (at the end of the test) 1.65 ± 0.14 0.14 ± 0.02

At the end of the test the SPSI of the juvenile trout from both groups were by 65 % higher than at the beginning, which might have to do with the influence of a multitude of factors on the immune system of the fishes kept in the aquaria [Latremouille, 2003].

It should be noted that the short-term alimentation with the extruded feed with LAB did not affect the external condition of the juvenile trout’s internals. The clinical checkup and the ichthyopathological examination of the fish did not reveal any typical signs of infectious and bodily diseases. The normal physiological condition of the juvenile trout in the test environment is confirmed by the data of analyzing the cellular composition and hematological

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parameters of the bloodstream, including the description of the cell morphology in health and disease and the registration of the erythrocyte sedimentation rate. The microscopy of blood films of P. mykiss from the test and the control group allowed obtaining the blood formula data exposed in Table 7. The erythrocytes were found out to be the most numerous elements in the bloodstream of the trouts from both groups; the respective fractions of erythrocytes were 65.20 % or 19.84 g/l and 74.60 % or 23.17 g/l (р0.05).

Table 7. P. mykiss blood formulae in the test and the control group

Group

Formed elements

erythrocytes

Immature lymphocytes

mature

lymphocytes monocytes granulocytes Blood disks leucocytes

g/l % g/l % g/l % g/l % g/l % g/l %

Test 19.84

±0.32 65.2 2.1

±0,16 8.6 4.41

±0.24 19.7 0.71

±0.09 2.02 0.52

±0.09 1.38 0.9

±0.1 3.1

Control

23.17

±0.41 74.6 3.48

± 0,21 15.2 1.62

±0.14 5.9 0.42

±0.08 0.6 0.31

± 0.09 0.9 0,8

±0.1 2.8 The dominant component of the blood in the test group trouts was immature erythrocytes that were similar in appearance and ultrastructure as oval, flattened cells with eosinophilic cytoplasm and a centered elliptical nucleus (Fig. 2а); the nuclei were found out to contain both, heterochromatin and euchromatin; according to the test findings, that pointed to the functional activity of the given kind of blood cells [Kuchareva, 2019]. It should also be noted that some of the erythrocytes in the blood of the control group trout had abnormal shapes, such as poikilocythemia, with the occurrence rate of 16,6% (Fig. 3а), teardrop cells, with the occurrence rate of 3.2% (Fig. 3b), and nuclear introsusceptions, with the occurrence rate of 10.3% (Fig. 3c).

The blood stream of the trout from both groups appeared to contain a heterogeneous group of lymphocytes of varied maturity and, therefore, with different morphofunctional features. The aggregate fraction of lymphocytes in the test group trout was 31.7%, and mature lymphocytes were by 130 % more numerous than their immature counterparts. In the control group of P. mykiss the fraction of lymphocytes relative to blood’s other formed elements was 22.6 % and mature lymphocytes were by 150 % more numerous than their immature counterparts. The highest content of monocytes and granulocytes was registered in the blood of the test group trouts --- 2.02 % or 0.71 g/l and 1.38 % or 0.52 g/l, respectively. The fraction of blood disks in the blood of the test group trouts was by 0.3 % higher than for the control group trouts.

a b

Figure 2. Erythrocytes in the blood of the test (a) and the control group of P. mykiss (b) (1000×)

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a b c

Figure 3. Abnormally shaped erythrocytes in the blood of the control group of P. mykiss: poikilocytosis (а), teardrop cells (b), nuclear introsusceptions (с) (1000×)

The erythrocyte sedimentation rate in the blood of both groups of rainbow trout was estimated as an indicator that not only pointed to the existence of an inflammatory process in the body but also showed the condition of homeostasis in the test environment. For the dynamics of the changes in the ESR in both groups see the Boxplot in Fig. 4.

Figure 4. ESR sedimentation rate distribution in the test (a) and the control group of P. mykiss (b)

The ESR measurements revealed some differences in the ESR changes between the groups of Parasalmo mykiss. The ESR range of the control and the test group extended from 2.36 to 4.22 and from 2.67 to 4.09 ml/h, respectively.

Thus the respective average ESR were 3.57 and 2.92 ml/h. Both indicators are close to the standard rate for trout, though the ESR in the control group was by 20 % higher than in the test group. According to the data from the Boxplot in Fig. 4, the ESR of the test group trouts were less variable than the ESR of the control group trouts, despite the two extreme values of 2.09 (upper limit) and 4.09 ml/h (lower limit). The results can be explained by the fact that the availability of an additional immune stimulant in the form of L. brevis made the juvenile trout from the test group more sensitive to environmental factors, which affected the homeostasis of the fish organism and the ESR change to acceptable variations.

The changes in the activity of the cell factors of inherent immunity, that are responsible for the autarcesis of the juvenile trouts from both groups, are represented in Table 8 and in the shots of the phagocytosis pattern of the test (Figs. 5A and 5b) and the control group of P. mykiss (Fig. 5c).

Table 8. Average phagocytic activity of the leucocytes in the test and the control group of P. mykiss (р< 0.05) Phagocytic activity Phagocytic index Phagocytic intensity

Test group of P. mykiss

63.4 ± 5.2 3.11 ± 0.7 3.44 ± 0.85

Control group of P. mykiss

48.7 ± 3.75 2.17 ± 0.4 2.39 ± 0.65

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The blood of the control group trout contained an increased number of leucocytes absorbing from one to three cells of a phagocytosis object, and the phagocytic index in that group was 3.11 ± 0.70, whereas in the control group it was 2.17 ± 0.4. It should be noted that the fraction of actively phagocytic leucocytes was higher in the trouts alimented with the feed with LAB (3.44 ± 0.85 %) than in the control group (2.39 ± 0.65).

Figure 5. Phagocytosis pattern of the P. mykiss from the test (a, b) and the control group (c) of (1000×) For the results of studying the taxonomic diversity of the microbial background in the content of the examined trouts’

intestine see Table 9. The discovered microorganisms are referred to four types or phylums, including Firmicutes, Bacteroidetes, Actinobacteria и Proteobacteria. In all of the samples the greatest biodiversity was found in gammaproteobacteria represented by three orders, six families, and seven genuses.

Table 9. Taxonomic composition of the background flora of the intestine of P. mykiss Taxonomic categories

Type Class Order Family Genus

Firmicutes Bacilli Вacillales Bacillaceae Bacillus

Staphylococcaceae Staphylococcus Lactobacillales Lactobacillaceae Lactobacillus Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Cytophaga

Flexibacter Actinobacteria Actinobacteria Micrococcales Micrococcaceae Micrococcus

Propionibacteriales Propionibacteriaceae Propionibacterium Proteobacteria Betaproteobacteria Burkholderiales Alcaligenaceae Alcaligines

Gammaproteobacteria Enterobacterales Enterobacteriaceae Escherichia Klebsiella

Hafniaceae Hafnia

Morganellaceae Proteus Yersiniaceae Serratia Pseudomonadales Pseudomonadaceae Pseudomonas

The smallest biodiversity was elucidated for the taxons as part of β-proteobacteria (one genus – Alcaligines sp.) and bacteroids (two genuses – Cytophaga sp. and Flexibacter sp.). It should be noted that in all of the trouts from both

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groups pathogenic and opportunistic pathogenic bacteria were isolated to pure culture. The microorganisms identified according to Bergy’s manual represented group four (Alcaligenes and Pseudomonas), subgroup one of group five (Enterobacteriaceae fam. - Klebsiella and Proteus) and subgroup one of group 15 (Cytophaga and Flexibacter).

The findings about the biodiversity of microorganisms in the intestine of P. mykiss were used to analyze the dynamics of changes in the initial parameters of alimentation quality and balance as part of the microbial population upon the addition of LAB to the initial diet on the 10th and 20th day of the test. The job done is necessary for defining the criteria of applying LAB in the curative and preventive diet for trout with the control over the evolution of physiological and biochemical dysfunctions in the fish organism, followed by the derangements in the digestion and intake of nutrients as part of the used compound feed.

The distribution of the fraction of isolated microorganisms in the test and the control group is represented in standardized bar charts with the accumulation for comparison by categories of the percentage contribution of each taxonomic group to the general stabilization of the trout gut flora in the presence of LAB (Figs. 6 and 7). In the test group the fraction of Firmicutes, but for the representatives of Staphilococcus, increased by 5.3 % on average, whereas the fraction of LAB increased by 10.5 % on average. In the control group the fraction of Firmicutes fell by 7%, mainly due to a nearly fourfold reduction in the fraction of L. brevis. In the future, this change in the fraction of Firmicutes as part of the gut flora may weaken the biochemical activity with regard to hydrocarbons as part of the feed, and result in the deficiency of short-chain fatty acids. In addition, the reduction in the fraction of LAB in the control group is an alarm signal for stating the negative change, related to the protective function of the intestine walls, and suppression of anti-inflammatory cytokines. The number of Bacteroidetes in the test and the control group increased for the 20 days of the test by 10 and 50 %, respectively. Considering that the representatives of this type of bacteria populating the gastrointestinal tract play a leading role in the homeostasis control, it is necessary to take into account the probability of changes in their qualitative and quantitative diversity in the presence of L. brevis. In the test group the fraction of Actinobacteria, capable of suppressing the growth of pathogenic and opportunistic pathogenic species of bacteria and take part in the protection of the intestinal wall functions, increased from 0.8 % by the 10th day of the test to 3.5% by the 20th day of the test (Fig. 6).

In the intestine of the control group trouts placed on the supplement-free diet the fraction of such actinobacteria, as Micrococcus and Propionibacterium, also increased, from 1.6 to 3.8 %, though the difference did not exceed 130 %.

Proteobacteria include a broad range of pathogenic and opportunistic pathogenic gram-negative bacteria that represent such genuses, as Escherichia, Salmonella, Vibrio, Yersinia, Pseudomonas, cause mixobacterial diseases with diverse clinical implications, and are notable for covering many fish species, and resulting in complicated treatment of outbreaks

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Figure 6. Distribution in the fraction of separated microorganisms on the 10th and 20th day of the test in the test group of P. mykiss

[Sidorova et al., 2013]. With the inclusion of LAB in the diet for juvenile trout, the aggregate percentage content of proteobacteria fell by 160 %; plus, there was no release of Serratia and Proteus from the gut content on the 20th day of the test and the fraction of Alcaligines, Escherichia, and Klebsiella fell from 3.8 to 1.2 %. The qualitative and quantitative diversity of pathogenic and opportunistic pathogenic bacteria in the control group remained stable throughout the test and reached 31 to 36.4% (Fig. 7).

Figure 7. Distribution in the fraction of separated microorganisms on the 10th and 20th day of the test in the control group of P. mykiss

To impartially interpret the changes in the biodiversity of the microbial population of the trout intestine with the inclusion of LAB in the diet and analyze the numerical values of the basic properties of the background microbial population, we calculated such environmental biodiversity parameters, as Simpson’s (Simpson – 1-D), Shannon’s (Shannon – H), Menhinick’s (Menhinick – Dmn), Margalef’s (Margalef – DMg) indices and also Pielou's evenness index. The results were interpreted considering that 1-D and H were sensitive to the number of species in the gut flora but little sensitive to their size. DMn and DMg are more sensitive to the size of particular species of microorganisms. There are several features distinguished by the analysis of the enumerated biodiversity indices. In the surveyed microbial communities of the trout underyearlings from both groups Simpson’s index ranged from 0.91 to 0.96 (Fig. 8). In case of using the BioMar feed enriched with LAB the evenness of distributing the species by their abundance in the community was higher in the test group (0.61) than in the control group (0.52). Shannon’s index ranged from 3.7 in the test group to 4.01 in the control group. Menchinick’s and Margalef’s indices calculated for estimating the change in the biodiversity of the gut flora of the fish from both groups would exhibit significant variations, which was possibly due to the modulation of microbial population by the added LAB culture, to the nutritional adaptation of the species in the examined microbial community, and also to the quality and quantity of components in the BioMar feed used in the test. In addition to the specified factors, the gut flora can also be affected by the quality of the environment, stocking density, diet, alimentation methods, housing conditions, occurrence of pathogens, and age of fish. The highest values of DMn and DMg were found for the gut flora of the control group trouts fed with the BioMar compound feed without LAB (DMn = 11.22 and DMg = 21.9) (Fig.8). The findings show that the trout gut’s autochthonal flora is abundant with common species of microorganisms in both, the control and the test group, upon an increase in the fraction of eubacteria in the intestine of the trouts placed on the BioMar diet with LAB.

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Figure 8. Environmental biodiversity indices of the gut flora of P. mykiss placed on different diets

Conclusion

This study has allowed us to obtain the primary test data proving the applicability of the Lactobacillus brevis: 2k.Gv strain as a probiotic supplement to the curative and preventive feeds for trout. The fish breeding, hematological, and immunological parameters have been analyzed to estimate the influence of LAB on the morphophysiological condition of P. mykiss underyearlings. The comparative efficiency analysis of the consumption of the extruded BioMar feed with and without a dietary supplement to the basic diet of juvenile trout has allowed us to find out that the feeding ratio has been by 50 % below the control level, which confirms the higher efficiency of administering the feed with LAB. The feed with LAB has been found out to positively affect an increment in the absolute and relative fish weight by 1.97 g/day and 2.18 %, respectively, as well as the survival rate by 60 %. The curative and preventive diet administered to the trout did not cause any explicit clinical and ichthyopathological changes, and the HPSI and THE SPSI varied within normal limits.

As a result of our investigations and analyzing works [Zhiteneva, 2004; Davydov, 2006; Borovskaya, 2010], we have obtained the data confirming the validity of the hematologic parameters of the fish bloodstream as the impartial criteria of changes caused by environmental factors, including the pattern of the standard diet and its curative preventive alternative with LAB. As found out by analyzing the blood of the test and the control group of Parasalmo mykiss, the average ESR was 3.57 and 2.92 ml/h, respectively. With their fraction of 65.2 (test) and 74.6 % (control), erythrocytes have appear the most numerous elements in the blood of juvenile trouts. Mature erythrocytes have appeared to be the dominant kind of cells (69.4 %) in the blood of the test group trout; plus, that blood contained no abnormal cells of erythrocytic series. The fractions of mature, immature, and abnormal erythrocytes found in the blood of the control group fish were 49.1, 20.8 and 30.1 %, respectively. These differences can stem from alimentation conditions, changes in the quality of the life environment, and be determined by the activity of blood- forming tissue [Kukhareva, 2019]. The blood of the test group trout has appeared to contain by 40 % more formed leucocytic elements than the blood of the control group trout.

It has been found out that the cellular factors of the trout’s inherent immunity are stimulated in the presence of Lactobacillus brеvis due to a 30 % increase in phagocytic activity as compared with the trout placed in the supplement-free diet. The findings agree with the results of investigating the activity of the cellular factors of

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inherent immunity in the presence of LAB as immune stimulants. The amount of actively phagocytizing leucocytes in the blood of the trout fed with BioMar with and without LAB was within 3.44 ± 0.85 and 2.39 ± 0.65 % (p <0.05), respectively. Considering that leucocytes play the leading role in the development of the immune response and also in lipoprotein exchange [Ivanov et al., 2013], we can suppose that an increase in the fraction of leucocytes in the blood of the trout placed on the diet with LAB is a sign of the positive effect not only on immune reactivity but on metabolism as well.

The short-term introduction of Lactobacillus brevis: 2k.Gv to the diet of juvenile trout sufficed for successively stimulating their population in the intestine and the power of the immune system and reducing the fraction of pathogenic and opportunistic pathogenic infectious agents referred to Proteobacteria. The findings agree with the results of several studies of the influence of bacteria with probiotic potential on the rainbow trout organism [Brunt et al., 2007; Newaj-Fyzul et al., 2007]. When the P. mykiss underyearlings from the control group were fed with BioMar enriched with LAB, the latter remained viable as part of the microbial population of the fish’s gastrointestinal tract, survived the passage through the gaster, and remained in the intestine. The analyzed LAB culture can be assumed to remain metabolically stable in the aggressive environment fraught with enzymatic activity and a high feed digestion level, which improves the LAB’s probiotic potential. It should be emphasized that the enumerated properties of Lactobacillus brevis: 2k.Gv are the strain features of the species and can be optimized by changing the mode of cultivation, including the modification of the nutritional medium for ensuring the maximal output of efficient LAB mass.

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