Prosiding II International Symposium “Innovations in Life Sciences” (ILS 2020)

development of explants was observed on the medium with the addition of sucrose (Fig. 2). Table 1. Mean shoot height, number of leaves, and number of shoots explant of strawberry ‘Nashe podmoskovie’ under standard conditions Treatment Subcultures Mean 1 2 3 Shoot height (cm) A 2.11±0.06 2.96±0.12 6.45±0.28 3.84±0.15 B 1.59±0.02 2.74±0.08 4.10±0.12 2.81±0.07 C 1.63±0.03 2.38±0.08 6.11±0.42 3.37±0.17 control 3.34±0.14 3.92±0.12 6.79±0.32 4.68±0.19 Mean 2.17±0.06 3.00±0.11 5.86±0.28 - Least significant differences (Р < 0.05) 0.22 0.30 0.87 Number of leaves A 6.50±0.22 13.30±0.94 19.20±0.59 13.0±0.58 B 3.70±0.15 12.20±0.44 15.00±0.53 10.3±0.37 C 4.20±0.29 10.70±0.53 18.80±0.8 n.s. 11.2±0.54 control 6.90±0.31 16.60±0.42 20.00±0.66 14.5±0.46 Mean 5.32±0.25 13.20±0.58 18.25±0.66 Least significant differences (Р < - 0.05) 0.72 1.77 1.86 Number of shoots per explant A 2.10±0.23 3.10±0.23 4.20±0.20 3.10±0.22 B 1.40±0.30 2.20±0.24 2.70±0.21 2.10±0.25 C 1.50±0.22 1.80±0.24 4.10±0.23 2.50±0.23 control 2.80±0.20 3.20±0.20 4.00±0.21 3.33±0.20 Mean 1.95±0.23 2.58±0.22 3.75±0.21 Least significant differences (Р < - 0.05) 0.69 0.66 0.63 Table 2. Mean shoot height, number of leaves, and number of shoots explant of black currant ‘Bryanskiy Agat’ under standard conditions Treatment Subcultures Mean 1 2 3 Shoot height (cm) A 3.13±0.11 8.20±0.64 8.79±0.34 6.71±0.36 B 2.54±0.13 7.79±0.49 8.73±0.26 6.30±0.29 C 2.16±0.18 7.37±0.48 9.10±0.40 6.21±0.35 control 2.48±0.15 4.77±0.66 6.49±0.40 4.58±0.40 Mean 2.58±0.15 7.03±0.53 8.28±0.36 Least significant differences (Р < - 0.05) 0.41 1.50 1.01 Number of leaves A 4.60±0.22 9.00±0.21 9.20±0.32 7.60±0.25 B 3.90±0.23 9.60±0.31 9.20±0.32 7.57±0.28 C 3.80±0.32 8.50±0.26 9.80±0.29 7.40±0.29 control 4.60±0.22 7.40±0.52 9.10±0.31 7.03±0.35 Mean 4.23±0.25 8.36±0.35 9.32±0.31 Least significant differences (Р < - 0.05) 0.70 0.98 0.89 Number of shoots per explant A 1.70±0.33 1.80±0.25 3.80±0.25 2.43±0.28 B 1.60±0.27 1.70±0.21 2.60±0.16 1.97±0.21 C 1.30±0.21 1.90±0.28 3.20±0.29 2.13±0.26 control 1.50±0.31 1.30±0.26 2.10±0.18 1.63±0.25 Mean 1.52±0.28 1.68±0.25 2.90±0.70 Least significant differences (Р < - 0.05) 0.80 0.72 0.63 3 BIO Web of Conferences 30, 05004 (2021) https://doi.org/10.1051/bioconf/20213005004 ILS 2020

Table 3. Mean shoot height, number of leaves and number of shoots explant of black raspberry ‘Bryanskoe Divo’ under standard conditions Treatment Subcultures Mean 1 2 3 Shoot height (cm) A 1.73±0.05 2.36±0.05 2.97±0.14 2.35±0.08 B 1.93±0.03 2.29±0.09 3.53±0.23 2.58±0.12 C - - - - control 2.18±0.06 3.21±0.08 4.65±0.25 3.35±0.13 Mean 1.95±0.05 2.62±0.08 3.72±0.22 Least significant differences (Р < 0.05) 0.16 0.23 0.64 - Number of leaves A 5.40±0.40 6.20±0.25 6.40±0.37 6.00±0.34 B 6.60±0.26 5.80±0.35 7.20±0.25 6.53±0.28 C - - - - control 6.30±0.26 6.0±0.26 7.60±0.33 6.63±0.28 Mean 6.10±0.32 6.03±0.29 7.07±0.32 Least significant differences (Р < 0.05) 0.92 0.84 0.94 - Number of shoots per explant A 1.0±0.25 4.40±0.31 2.80±0.29 2.73±0.28 B 1.5±0.27 3.10±0.41 3.20±0.55 2.6±0.41 C - - - - control 2.0±0.29 5.20±0.42 3.60±0.31 3.6±0.34 Mean 1.53±0.28 4.23±0.38 3.20±0.40 Least significant differences (Р < 0.05) 0.82 1.10 1.17 - a (C) b (C) c (A) Fig. 2. Regenerating plantlets of plum evaluated after the third subculture under standard medium, temperature, and light conditions after cold storage (bar 10.0 mm). Genotypes: a strawberry variety ‘Nashe Podmoskov'e’, b raspberry cultivar ‘Bryanskoe Divo’, c black currant variety ‘Bryanskiy Agat’ examples were cold preserved in the Murashige-Skoog medium supplemented with (A) 0.45% of mannitol, 0,7 mg L -1 BAP, 0,1 mg L -1 IBA and (C) 3% of sucrose 0,7 mg L -1 BAP and 0,1 mg L -1 IBA. 4 BIO Web of Conferences 30, 05004 (2021) https://doi.org/10.1051/bioconf/20213005004 ILS 2020

4 Conclusion Morphometric indicators of the development of regenerated plants on a nutrient medium after the action of mannitol with different concentrations differed depending on the species and variety. It was established that in all studied berry crops, along with the growth of the main shoot, a new shoot formation was observed. The most active formation of adventitious shoots was noted in the raspberry variety ‘Bryanskoe Divo’, which formed an average of 5.20±0.42 shoots per explant in the second subculture. At the same time, shoots with several nodes developed that could be cut for further propagation. When studying the effect of the reproduction cycle on the morphogenesis of meristemic cultures, it was found that the dynamics of changes in shoot length and number of leaves depended on the concentration of the active substance. So, for example, the shoot length at different concentrations of mannitol for black currant variety 'Bryanskiy Agat' by the second passage of development was 8.20±0.64 cm on nutrient medium A and 7.37±0.48 cm on medium C, and the shoot length increases to 8.79±0.34 and 9.10±0.40 cm, respectively. In variant C of the experiment, the maximum number of leaves was also noted. References 1. P. W. Jackson, K. Kennedy, Trends Plant Sci. 14, 578 (2009) 2. Gene bank standards for plant genetic resources for food and agriculture: Second edition, corrected and amended. (FAO, Rome, 2015). 3. N. I. Dzyubenko, Biopreserv. Biobanking, 16(5), 377 (2018) 4. F. Engelmannn, In Vitro Cell. Dev. Biol. Plant 47, 5 (2011) 5. M. Lambardi, C. Ruta, “Biotechnology for plant genetic resources conservation: an overview of in vitro-banking and cryobanking in the world”, in the VIII International Scientitific and Practical Conference «Biotechnology as an Instrument for Plant Biodiversity Conservation (physiological, Biochemical, embryological, genetic and legal aspects)», p. 15, 2018. 6. D. Shah, N. Sajjad, R. Ali, N. Nazir, S. Hassan and S. Shah, Plant Signaling Molecules, 257–268 (2019) 7. A. Lisek, T. Orlikowska, Acta Hortic., 560, 189 (2001) 8. A. Lisek, T. Orlikowska, Biotechnologia 3, 237 (2001) 9. A. Vinoth, R. Ravindhran, IJCOA 02, 268 (2013) 10. A. Lisek, T. Orlikowska, PCTOC 78, 167 (2004) 11. H. T. Gomes, P. M. C. Bartos, M. de A. Teixeira, R. F. Almeida, L. de L. Florencio, J. E. Scherwinski-Pereira, Pesq. agropec. bras., 52(12), 1286 (2017) 5 BIO Web of Conferences 30, 05004 (2021) https://doi.org/10.1051/bioconf/20213005004 ILS 2020

Biotechnological potential of fungi and bacteria with ligninolytic activity (mini-review) Yulia Kurkina, Vasili Travkin, and Inna Solyanikova* Belgorod State National Research University, Belgorod, Russia Abstract. The presented mini-review gives a general idea of oxidative enzymes of fungi and bacteria. Possible directions of their practical application are shown. 1 Introduction A huge amount of pollutants enters the environment as a result of industrial development, urbanization and intensive agriculture with the simultaneous use of a large number of pesticides. In recent years, more and more attention has been paid to the development of and the introduction of resource-saving technologies and increasing the degree of environmental safety of industrial enterprises through the further use of various natural nutrient substrates. Lignin, the second after cellulose naturally occurring compound, is a highly crosslinked polymer formed by the polymerization of 4- hydroxyphenylpropanoid monomers (monolignols) via ether and carbon-carbon bonds. Phenolic fragments of monomer units are p-hydroxyphenyl, guaiacyl and syringyl groups, and the percentage of them depends on the type of plant and tissue (Fig. 1). Lignin can be considered not only as a pollutant of paramount importance. In an expanding bioprocessing industry, in which large quantities of carbohydrate biomass are subsequently used to produce chemicals, further valorization of lignin is an essential part of the processing process. Depolymerization of lignin leads to the formation of a mixture of monomeric and oligomeric aromatic compounds. The use and conversion of these lignin-based aromatic compounds will pave the way to get various products in biorefineries [1, 2]. Lignocellulosic plant wastes from agriculture and the woodworking industry are the dominant type of potential plant material for microbiological conversion. When using biological agents, plant waste can be processed into valuable target products while reducing the rate of environmental pollution. The ability of microorganisms to destroy the lignocellulosic complex formed from plant waste is not the same in terms of the duration and processing depth. One of the promising biological agents producing ligninolytic enzymes are micro- and macroscopic fungi [3]. As for bacteria, in recent years the number of publications showing a high potential of this group of microorganisms in the destruction of ligno-cellulose residues has increased significantly. The purpose of this work is to evaluate the possibility of usage bacteria and fungi in biotechnologies for cleaning the environment from ligno-cellulose residues. 2 Material and Methods The main materials of this review were obtained as a result of a study of literary sources. 3 Results and discussion Due to chemical bonds and many aromatic residues, lignin is compound highly resistant to a degradation [4]. The lignin degradation enzymes of white rot fungi are most fully studied [5]. Lignin destruction is initiated by peroxidases and / or laccases. Under the action of peroxidases on lignin, its oxidative fragmentation occurs with the formation of polyphenolic products. The formation of dimers occurs during the oxidation of phenolic monolignols to their corresponding phenolic radicals. Along with this, there are processes of condensation of lignin. Reverse polymerization is facilitated by subsequent enzymatically-catalyzed oneelectron oxidations [6, 7]. Condensed lignin containing C—C diphenyl structures exhibits high resistance to enzymes, which leads to the formation of chains of dark granules, and this is the reason for the appearance of a “marble” pattern on wood [8]. There is an opinion that peroxidase has a wider substrate spectrum than other enzymes of the lignolytic complex [9]. It has been established that they use various types of hemecontaining peroxidases, which include the so-called lignin peroxidases (LiP), manganese peroxidases (MnP), universal peroxidases (VP) and peroxidases, bleaching dyes (DyP) [10]. Lignin peroxidase (EC 1.11.1.14) is a heme-containing glycoprotein that catalyzes the cleavage of C–C bonds in polymer lignin molecules, the oxidation of benzyl alcohols and methyl groups in benzyl *Corresponding authors: [email protected] © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 05005 (2021) https://doi.org/10.1051/bioconf/20213005005 ILS 2020

compounds, and the opening of an aromatic ring [11]. Manganese peroxidase (EC 1.11.1.13) is a glycoprotein containing heme as a cofactor. The enzyme oxidizes Mn2+ ions to Mn3+ in the presence of hydrogen peroxide, which, in turn, oxidize phenol derivatives, hydroxylate aromatic compounds [12]. Fig. 1. The structure of lignin The lignolytic complex of basidiomycetes also includes phenol oxidase laccase (paradiphenol: oxygen oxidoreductase, EC 1.10.3.2). Laccase is a coppercontaining glycoprotein that catalyzes the oxidation of polyphenols and polyamines, some inorganic ions in the reaction of reduction of molecular oxygen to water [13]. Laccases are capable of catalyzing the one-electron oxidation of organic compounds to the corresponding radical species. Laccases in the catalytic center contain a cluster of 4 copper ions, oxygen serves as an electron acceptor, with the formation of water as a by-product. The resulting radical products may undergo further oxidation, hydration, disproportionation, or polymerization. The work of the enzyme leads to the formation of phenoxy radicals and quinones, which initiate spontaneous reactions, including the breaking of C–C bonds in aromatic substrates, their demethoxylation, demethylation and polymerization, as well as the formation of reactive oxygen species. Unlike ligninolytic peroxidases, which are characteristic exclusively of white rot basidiomycetes and their litter fungi, laccase is also produced by ascomycetes, deuteromycetes, bacteria and is the most common oxidative enzyme in soils [14]. Information about the catalytic properties of fungal laccases, their isoenzyme composition is known, but only for limited genera [15]. The most thoroughly studied are the oxidoreductases of basidiomycetes [16], while the literature on the deuteromycete enzymes is still contradictory and the number of fungi studied in this 2 BIO Web of Conferences 30, 05005 (2021) https://doi.org/10.1051/bioconf/20213005005 ILS 2020

direction is small. It is also known that the vital activity of imperfect fungi is crucial for the initial stages of the biodestructive process and the preparation of material components for utilization by other groups of destructive organisms. It was established that the maximum activity of Aspergillus terreus and Trichoderma viride peroxidase, phenol oxidase, and catalase in most cases is observed on days 10–13, while the pH of the medium optimal for the manifestation of enzyme activity is in the range 7.2–8.2 [17]. Micromycetes are natural active destructors of wood lignin; phenol oxidase of microscopic fungi from the genera Trichoderma and Fusarium are involved in biodegradation of lignin [18, 19]. It is known that microscopic fungi, in comparison with bacteria, exhibit the highest exoenzymatic activity and use up to 60% of the substrate to build their cells, while in bacteria its utilization rate is on average 35% [20]. It should be noted that only xylotrophic fungi are able to decompose lignin, possessing a complex of oxidase and hydrolase enzymes. The decomposition of all components of the lignocellulosic substrate can occur almost simultaneously or with the predominant decomposition of lignin, while the cellulose remains relatively unaffected, which depends on the sequence of induction of certain enzymes [21]. Intensive delignification of hardwood is accompanied by the active consumption of xylose, which is part of the glucuronoxylan hemicellulose complex. To decompose lignin, a cosubstrate is needed, which some hemicelluloses can serve. A characteristic of the destructive ability of xylotrophic fungi is the loss of wood caused by them in the laboratory. For 196 days, the fungus may be able to destroy spruce wood by 54%, and aspen by 58%. The decomposition of lignin by xylotrophic fungi is directly proportional to the loss of wood mass and has a linear relationship [22]. It is known that it is advisable to search for promising strains for the destruction of plant residues among soil micromycetes [23]. Thus, strains of Trichoderma koningii, Ulocladium botrytis, Curvularia lunata, Alternaria alternata, and Bipolaris australiensis with high values of lignin degradation ability were isolated from the rhizosphere of leguminous plants [23, 24]. In contrast to the relatively well-studied fungal ligninolytic complex, the ligninolytic ability of bacteria still need to be studied. In recent years, a growing number of articles describes bacterial lignolytic enzymes. Typical lignin peroxidases characteristic of fungi were not found in bacteria. Perhaps this is due to the peculiarities of the expression of these rather complex glycosylated proteins containing several disulfide bonds and including several calcium ions and a heme cofactor [6]. However, studies have shown that bacteria possess a specific type of peroxidase called bleach dyes peroxidase (Dy-P) [25]. The first Dy-P was described in 1999, but only recently it has been shown that this enzyme is widely distributed in bacterial genomes [10]. Dy-P was found in Escherichia coli K-12, Thermobifida fusca YX, Rhodococcus jostii RHA1, Streptomyces viridosporus strain T7A, Streptomyces coelicolor A, Amycolatopsis sp. 75iv2, Pseudomonas sp. strain YS-1p. Depending on the sequence, Dy-Ps in the database are divided into four classes [26]. Enzymes belonging to classes AC are mainly found in bacteria, while members of the class D-DyPs are extracellular representatives of fungi [27]. Class A Dy-Ps typically have a Tat signal sequence, i.e. are secreted. The protein sequences of class B and C Dy-Ps are intracellular enzymes. The InterPro database currently contains 8318 Dy-P sequences. But only about thirty of them were isolated and characterized [27, 28]. Dy-Ps are mainly active at acidic pH and exhibit a very broad substrate profile, including several classes of synthetic dyes, monophenol compounds, veratril alcohol, β-carotenoids, Mn2+ and lignin model compounds, but their physiological substrates remain unknown. In general, the bacterial DyPs studied to date have a lower oxidative ability than fungal analogues, are usually active with monophenol substrates, but some bacterial Dy-Ps show significant activity against non-phenolic veratril alcohol. Some bacterial Dy-Ps are capable of oxidizing 4- methoxymandelate in the presence of Mn2+ and O2, without requiring the presence of H2O2, acting as oxidases. Very promising is the Dy-P of strain Pseudomonas fluorescence Pf-5, capable of modifying alkaline kraft lignin, a by-product of paper production. This is the most stable bacterial peroxidase (60% residual activity after incubation at 70 ° C for 2 h) with high stability in alkaline solutions (> 80% activity after incubation at pH 5-10 at 37 ° C for 1 h) [29]. The second type of ligninolytic enzymes is ligninmodifying laccases, including bacterial ones. Like fungal laccases, many bacterial laccases are secreted and usually carry out polymerization or depolymerization reactions. The Tat secretion system is used to export bacterial laccases. Since laccases do not need cofactors and do not form toxic H2O2, they are biotechnologically significant biocatalysts, unlike many oxidoreductases. The greatest number of laccases is characterized in representatives of the genus Streptomyces. Laccases are also known in bacteria of the genera Bacillus and Thermus. Laccases are used in bioremediation processes for disinfecting industrial wastewater, in the food industry for stabilizing drinks and improving the organoleptic properties of food products, in the synthesis of pharmaceuticals and other fine chemicals, in the manufacture of textile dyes and in the delignification of wood, especially in the bleaching process [30]. Laccases can also be used in pretreatment of coniferous species to improve subsequent hydrolysis. Laccases exhibit high potential in biotransformation of various pollutants. These enzymes are used as crude extracts of enzymes, free or immobilized enzymes. Application of laccase in bioremediation and water treatment on a large scale are limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery, and utilization. These problems also associated with operational problems and the need for large-scale production of varnished products [31]. Therefore, based on data on the characteristics of water bodies that need to introduce treatment 3 BIO Web of Conferences 30, 05005 (2021) https://doi.org/10.1051/bioconf/20213005005 ILS 2020

technologies, it is necessary to identify and develop new laccases that can be used in difficult conditions and develop new strategies and processes to achieve their effective application in the treatment of contaminated water. Recently, the problems of stability, efficiency, separation and reuse have overcome the immobilization of enzymes and the development of new biocatalytic materials. 4 Conclusion For the successful use of lignolytic enzymes in industry, of particular interest are more stable enzymes with high catalytic activity. The search for such enzymes is of the great interes. Three strategies can be mentioned for the search for microorganisms with the ability to decompose lignin. The first one is the isolation, characterization and verification of each individual strain. In the Table the most perspective strains are listed. As an example, gene encoding a laccase from a thermophilic aerobic bacteria Caldalkalibacillus thermarum TA2.A1 was cloned and expressed in E. coli [32]. It was found that laccase from this strain was monomeric protein with a molecular weight of 57 kDa. Table 1. Microbial strain capable of lignin degradation and their biotechnology application Strain Enzyme Potential application Peculiarities Ref Collybia dryophila Mn-Peroxidase Humus turnover by recycling of high molecular-mass humic substances. Manganese peroxidase (MnP) was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. The production of MnP was noticeably increased in Mn2- supplemented cultures. [33] Trametes versicolor and Trametes villosa Laccase Decolorization of different soil fulvic acids The ability of laccases from Trametes species to transform soil FAs was shown for the first time [34] Trichoderma viride and Trichoderma lignorum Coppercontaining oxidoreductases Soil, rizosphere of plants Lignin degradation [35] Cladosporium herbarum Peroxidase Soil, rizosphere, phyllosphere [36] Phellinus igniarius Peroxidase White birch wood Under the action of peroxidases on lignin, its oxidative fragmentation occurs with the formation of polyphenolic products. In parallel with this, lignin condensation processes take place: [8] Funalia trogii Laccase Wood mushrooms Laccases from basidiomycetes are characterized by a high redox potential and broad substrate specificity [37] Bjerkandera adusta Phenol oxidase Inhabit the bark of trees Extracellular peroxidases are regulated [21, 37] Aspergillus terreus and Trichoderma viride Peroxidase phenol oxidase wood The activity of extracellular peroxidase, catalase and phenol oxidase of fungi is regulated by some chemical compounds (СuSO4, fungicides “Caton LXE” and “Ecodes”). [17] Acremonium murorum and Botrytis cinerea Laccase Gray forest soil and black soil Humic acids have a mixed effect on laccase products [14] Fusarium culmorum, Fusarium sporotrichioides, Fusarium solani and Trametes versicolor Laccase, MnPeroxidase, lignin peroxidase Forest soil and tree bark Strains secreted laccase, manganese peroxidase and lignin peroxidase into the growth medium under butch cultures; the yield of enzymes in the culture medium averaged from 75.5% to 91.9%, which facilitates their isolation and purification [19] Anoxybacillus sp. strain UARK-01 Laccase Thermophilic bacterium from soil. The strain grows optimally in the presence of oxygen at 55 °C and pH 8. The laccase activity was optimal at 90 °C and pH 9 [38] 4 BIO Web of Conferences 30, 05005 (2021) https://doi.org/10.1051/bioconf/20213005005 ILS 2020

The second approach is based on the emergence and development of new biotechnologies and bioinformatics tools that allow the search for new enzymes using the metagenome. These approaches can make a significant contribution to the future economical production from renewable resources [39]. The third approach is the creation of highly efficient multiple analysis and strain search systems with specified characteristics, performed by the newly created enzyme systems. One of such highthroughput screening strategy for searching alkali lignindegrading microorganisms developed by Chong et al. [40] is based on the ferricyanide-based assay involving the formation of Prussian blue. The use of this screening system made it possible to isolate from soil the strain Rhodococcus pyridinivorans CCZU-B16, capable of degradation 30.2% of 4 g/L alkali lignin under the nitrogen-limited condition at 30 °C for 72 h. In addition, a distinctive feature of this strain was the accumulation of lipids in the cells. Among the C16 and C18 fatty acid profiles, four major ones prevailed, including palmitic acid, palmitoleic acid, stearic acid and oleic acid [40]. Based on the data obtained by various scientists studying the ability of microorganisms to transform lignin, we can conclude the high potential significance of such microorganisms for different areas of biotechnology, e.g. to clean the environment of lignin, alkali lignin resulting from the pulp and paper industry, for processing plant residues of agricultural waste, to obtain high-value compounds from lignin. In light of the data obtained by various scientists studying the ability of microorganisms to transform lignin, we can conclude the high potential significance of such microorganisms for different areas of biotechnology, e.g. pulp and paper industry, textile, food industries, bioremediation, organic synthesis, pharmaceutical sector, nanobiotechnology, a biofuel cell, and processing of agricultural waste [41]. Acknowledge This work was carried out in the framework of the program Scientific and Educational Center "Innovative solutions in the agro-industrial complex", Belgorod. The authors thank the staff of the Department of Biotechnology and Microbiology and students A.S. Barskovoi, E.P. Yesina and Yu.V. Gushchina for technical assistance in conducting a series of laboratory experiments. References 1. O.Y. Abdelaziz, D.P. Brink, J. Prothmann, K. Ravi, M. Sun, J. García-Hidalgo, M. Sandahl, C.P. Hulteberg, C. Turner, G. Lidén, M.F. GorwaGrauslund, Biotechnol. Adv. 34(8), 1318 (2016) 2. A.J. Ragauskas, G.T. Beckham, M.J. Biddy, R. Chandra, F. Chen, M.F. Davis, B.H. Davison, R.A. Dixon, P. Gilna, M. Keller, P. Langan, A.K. Naskar, J.N. Saddler, T.J. Tschaplinski, G.A. Tuskan, C.E. Wyman, Science 344(6185), 1246843, (2014) 3. T.V. Ryazanova, N.A. Chuprova, T.A. Luneva, in Catal. Ind. 6, 64 (2014) 4. A.M. Abdel-Hamid, J.O. Solbiati, I.K.O. Cann, Adv. Appl. Microbiol. 82, 1 (2013) 5. E.V. Dorshakova, N.P. Elinov, A.N. Mamoshin, Problems of Medical Mycology 16(4), 41 (2014) 6. G. de Gonzalo, D.I. Colpa, M.H.M. Habib, M.W. Fraaij, J. Biotechnol. 236, 110 (2016) 7. P. Muradov, Sh. Gasymov, F. Gahramanova, A. Alieva, D. Abbasova, Sh. Babaeva, M. Ragimova, Vestnik MGOU, 1, 109–112 (2009). 8. G.N. Kononov, A.N. Veryovkin, N.L. Goryachev, S.S. Nikitin, Lesnoi vestnik 6, 112 (2015) 9. V.V. Shutova, V.V. Revin, and T.V. Kudaeva, in Isvestia of Saratov University, part Chemistry, Biology, Ecology, 13, 80–85 (2015). 10. C. Lambertz, S. Ece, R. Fischer, U. 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Yakovleva, Vestnik OrelSAU 3(24), 36 (2010) 19. I.V. Darmov, E.I. Gorshunova, T.S. Tarasova, Scientific notes of the Kazan University. Natural Sciences Series, 159(1), 72 (2017) 20. I.S. Sandanova, “Microbiological destruction of plant litter of steppe ecosystems of southeastern Transbaikalia”: research, 2007: 127. 21. I.A. Kazartsev, V.I. Roshchin, VA. Solovyov, Mycology and phytopathology 48(2), 112 (2014) 22. K.A. Vyrastkova, I.G. Wide Adv. Sci. 1, 7 (2017) 23. Yu.N. Kurkina, A.S. Barskova, and E.P. Esina, “Study of kinetics of micromycetes, which produced lignolytic enzymes in vitro” in “Current status, problems and prospects for the development of agricultural science: materials of the IV International Scientific and Practical Conference, Yalta, sept. 2019. 5 BIO Web of Conferences 30, 05005 (2021) https://doi.org/10.1051/bioconf/20213005005 ILS 2020

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Introduction of hyssopus officinalis l. into in vitro culture to optimize the conditions for obtaining callus tissues and microclonal propagation as a promising metod of innovative agrobiotechnologies Elena Maslova1* , Natalya Gulya2 , Tatyana Perelugina1 , Valeria Semykina1 , and Elena Kalashnikova3 1Belgorod State National Research University, Belgorod, Russia 2Biotech company «Biocad», Moscow, Russia 3Russian State Agrarian University – Moscow Timiryazev Agricultural Academy Abstract. The sterilization process of plant explants of H. officinalis was optimized when introduced into an in vitro culture, the most effective sterilization modes, optimal sterilizing agents, their exposure time and concentration were selected. Callus tissues and mini-plants of H. officinalis were obtained in vitro and the most optimal nutrient media were determined both for microclonal propagation and for the induction of callus tissue H. officinalis, which can be further used for mass cultivation of cell and culture and obtaining safe bio-additives with active substances for livestock and crop production as a part of the development of modern agrobiotechnologies. 1 Introduction Modern agrobiotechnology is currently the main priority area of development of the Russian Federation. To increase the efficiency of crop production and obtain greater productivity and yield of the target product, innovative agricultural and biotechnological methods are used. So, for example, microclonal propagation is used for further mass propagation and cultivation of highquality healthy plants by the traditional method in field and greenhouse conditions. The same method of cell and tissue culture can be used to obtain callus tissue of plants and to cultivate their cellular plant biomass in laboratory conditions and bioreactors in industrial biotechnological enterprises to obtain the target product, containing valuable biologically active substances or components with antibacterial, antimicrobial, antitumor and immunomodulating properties. They can be used in animal husbandry as a biological supplement and in crop production as a biological product that reduces the use of chemical additives and drugs that increase the immunity of both plant and animal organisms. One of such new promising sources is the representative of the Lamiaceae family - Hyssopus officinalis L. is a representative of the genus Hyssopus. H. officinalis is a perennial semi-shrub plant that grows under natural conditions in the southern regions of the Russian Federation, the Caucasus, the Black Sea region, the Crimea, Altai, Central Asia, Europe and North Africa [1-2]. H. officinalis is a honey plant, as well as a medicinal and a spicy aromatic plant. It is included in the pharmacopoeias of France, Sweden, Germany, Romania, and Portugal [3]. It contains essential oil, sesquiterpenes, tannins, oleanolic and ursolic acids, resins, mineral salts, organic acids, flavonoids [2, 4]. It is known that in the flowering phase, the aerial mass of H officinalis contains up to 1.5% of essential oil in terms of dry weight; flavonoids (apigenin, luteolin, quercetin), their glycosides and phenolic acids (chlorogenic, protocatechuic, ferulic lilac, phydroxybenzoic coffee, vanillin, p-coumaric, rosmarin and gentisin); Vitamin C and carotene, along with many other substances [5-7]. The essential oil of H. officinalis, which is obtained in many countries of the world, is of great interest [8-9]. The chemical composition of H. officinalis essential oil was studied in various parts of the world, and according to the results of most studies, the main components are pinocamphone and isopinocamphone [5–7, 10-13]. The composition of the essential oil and the quantitative content of various compounds differ depending on soil-climatic and genetic factors [13]. Nevertheless, the main components characteristic of this plant are pinocamphone, isopinocamphone, β pinene, sabinene, myrcene, βfellandren, linalool, myrtenol, elemol and germacren-D. Thymol and methyl acetate chemotypes of H. officinalis also grow in Iran [14-15]. Hyssopus vegetable raw materials and essential oils are used in perfumes and cosmetics (aromatic components of perfumes, cosmetics, soaps), food (spice and fragrant seasoning for flavouring cold snacks, meat and fish dishes, sauces, alcoholic beverages) and the pharmaceutical industry. Hyssopus has excellent phytoncidal properties [16-17]. *Corresponding author: [email protected] © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

Of particular value are the leaves of the plant. Hyssopus officinalis has anti-inflammatory, antiseptic, antitussive effects. It stimulates digestion and relieves spasms of the smooth muscles of the gastrointestinal tract [1, 18]. Hyssopus infusions are used medicinally for diseases of the upper respiratory tract, cough, bronchitis, bronchial asthma, pneumonia and pulmonary tuberculosis, angina pectoris, neurosis, rheumatism, polyarthria, for the treatment of bruises, wounds, skin diseases [1]. This species has an expectorant, antispasmodic, decongestant, tonic effect, and some of its species exhibit a strong antiviral effect, especially against the herpes virus [18]. In experiments, conducted by scientists, Hyssopus officinalis exhibits antibacterial, antimycotic and antiprotozoal activity [19], and also has fungicidal properties [20]. Overall, it is clear that this culture is increasingly attracting the attention of scientists, due to the accumulation of a large number of biologically active compounds in plant tissues, which has been used in the pharmacy and food industry, and can also be used in modern agrobiotechnologies. Therefore, the relevance of studying of H. officinalis in an in vitro culture is evident and it is one of the priority areas of modern agrobiotechnology. To date, data on the study of the influence of cultivation conditions on clonal micropropagation and on the callus and morphogenesis of H. officinalis are not numerous [21-24], and the optimization of the in vitro cultivation process and the detailed study of this plant for the purpose of agrobiotechnology remains an urgent task. The aim of our study is to optimize the sterilization process and the conditions for obtaining callus tissues and microclonal propagation as a promising culture for use in modern agrobiotechnologies. 2 Experimental For research, the plant material of intact H. officinalis plants growing on the territory of the Botanical Garden of the National Research University “BelSU” was collected. To introduce into in vitro culture and to obtain seedlings and mini plants, plant seeds were used as plant explants. Sterilization was carried out in a stepwise manner according to generally accepted methods [25], using various disinfectants. As sterilizing solutions, we used: sodium hypochlorite in a concentration of 5-15% and 2.5-7.5%; lysoformin 3000 - 3%, 5% and 10%; biocide - 3%, 5% and 10%; silver nitrate - 0.1% and 0.5%; chloramine B at a concentration of 5% and 10%. Sterilizing solutions were left to expose to plant explants for 10, 20 and 30 minutes. At first, the plant material, washed in a soap solution, was placed in 70% ethyl alcohol for 1 minute, and then in a sterilizing solution of a certain concentration and for a certain time and after that it was washed three times in sterile distilled water. Sterile seeds were placed on the hormone-free Murashige-Skoog nutrient medium [26]. Further cultivation was carried out on a modified agar Murashige-Skoog medium [26] with a different composition of phytohormones (Table 1). Nutrient media were made according to generally accepted in vitro culture methods for plant cells and tissues (the hormone-free Murashige-Skoog medium was used as a control) [27]. All work and manipulations with cultures were carried out under aseptic conditions in the Lamsystems laminar box of protection class II, type A2, according to generally accepted sterility practices when working with cell and tissue cultures [27-28]. After seed germination the seedlings were transferred, under aseptic conditions, to nutrient media containing various concentrations of phytohormones and cultivated in a light room at a temperature of 23 ° C with observance of the day / night regime: 16/8 hours for 30 days. Table 1. Composition of modified Murashige-Skoog nutrient media Components of the medium MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 Volume per 1 L Macrosalt stock solutions, ml 100 100 100 100 100 100 100 100 Microsalt stock solutions, ml 1 1 1 1 1 1 1 1 Fe-chelate, ml 5 5 5 5 5 5 5 5 CaCl2, ml 5 5 5 5 5 5 5 5 Vitamins: РР, mg 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 B1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 В6 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Mesoinositis, mg 100 100 100 100 100 100 100 100 Glycine, ml 2 2 2 2 2 2 2 2 α-Naphthaleneacetic acid (NAA), mg - - - - - 0.5 2 - Indole-3-acetic acid (IAA), mg - - 1 - 2 - 2 3 Indole-3-butyric acid (IBA), mg - - - 1 - - - - Kinetin, mg - - - - 0.2 0.1 1 2 6-benzylaminopurine (6-BAP), mg 1 3 - - - 1 1 - 2,4-Dichlorophenoxyacetic acid (2,4-D), mg - - - - - - 2 - Sucrose, g 20 20 20 20 20 20 20 20 Agar-agar, g 7 7 7 7 7 7 7 7 2 BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

The quality of seedling growth in various nutrient media was assessed at weeks 1, 2 and 3 of cultivation using morphometric indicators such as shoot length in centimetres, average number of leaves in pieces, root length and the presence or absence of wilting and discoloration of leaves. Statistical data was treated using methods of mathematical statistics with the calculation of the arithmetic average, its error, and the Fisher criterion in Microsoft Office Excel [29]. 3 Results and discussion As a result of experimental work in vitro, optimal sterilizing agents of H. officinalis were determined and data was obtained on the effect of the most effective sterilizing agent and its sterilization mode. The effect of sterilizing agents on obtaining sterile and viable explants of H. officinalis during seed sterilization, as well as sterilization modes are presented in Table 2 and Figure 1. According to Fisher’s exact criterion, at a significance level of P> 0.05, in terms of its effect on the number of sterile explants, all the obtained data presented in the table significantly differ from the control. As it can be seen from table 2 and figure 1, the optimal sterilizing agents for H. officinalis seeds are a 5- 15% solution of sodium hypochlorite with its effect for 10 minutes and a 10% solution of chloramine B with its effect for 10 minutes, since under these conditions the maximum ratio of the number of sterile (100%) explants to the number of viable explants (100%) was obtained. It is also possible to sterilize seeds with 3% biocide for 30 minutes (86.0% viable explants), 0.1% silver nitrate with an exposure time of 10 minutes (80.33%). Sterilization with 10% chloramine B for 20 minutes, 5% chloramine B for 20 and 30 minutes and 5-15% sodium hypochlorite for 20 minutes gives the same yield of viable explants (60.67%). You can also use 5% lysoformin 3000 for 10 minutes (59.67% viable explants) and 5% biocide over time exposure time of 20 minutes (57.3% of viable explants). It is not advisable to use all other sterilization modes, since they produce less number of viable plant explants (less than 50%) or have a detrimental effect on the seeds. In the course of the work the selection of the optimal composition of nutrient media was carried out to obtain mini plants of H. officinalis L., and callus cultures in vitro. Plant sprouts of H. officinalis were cultivated on different media (the compositions are described in the research methods). The obtained parameters of the growth and development of mini-plants are presented in table 3, where SL is the shoot length, NL is the number of leaves, DR is the darkening of the root, CP is the color of the plant. Based on the obtained data, the optimal medium for obtaining mini plants of the H. officinalis species is MS5 medium, where there are more leaves, a more developed root system, and, in general, a more viable state of plants compared to other media. Optimum media for the induction of callus genesis are MS4 and MS6. Fig 1. The effect of sterilizing agents on the number of sterile and viable explants of H. officinalis 3 BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

Table 2. The effect of sterilizing solutions on plant explants H. officinalis Sterilizing solution and its concentration Sterilization duration (minutes) Sterile explants number (%) Viable explants number (%) Lysoformin 3000 (3%) 10 99.67±0.11 14.67±0.11 Lysoformin 3000 (3%) 20 100.0±0.0 0.0±0.0 Lysoformin 3000 (3%) 30 100.0±0.0 0.0±0.0 Lysoformin 3000 (5%) 10 100.0±0.0 59.67±0.11 Lysoformin 3000 (5%) 20 100.0±0.0 0.0±0.0 Lysoformin 3000 (5%) 30 100.0±0.0 0.0±0.0 Lysoformin 3000 (10%) 10 100.0±0.0 0.0±0.0 Lysoformin 3000 (10%) 20 100.0±0.0 0.0±0.0 Lysoformin 3000 (10%) 30 100.0±0.0 0.0±0.0 Biocide (3%) 10 100.0±0.0 21.0±0.2 Biocide (3%) 20 100.0±0.0 40.33±0.11 Biocide (3%) 30 100.0±0.0 86.0±0.2 Biocide (5%) 10 100.0±0.0 40.33±0.11 Biocide (5%) 20 100.0±0.0 57.3±2.3 Biocide (5%) 30 100.0±0.0 41.0±0.2 Biocide (10%) 10 98.0±0.2 0.0±0.0 Biocide (10%) 20 100.0±0.0 0.0±0.0 Biocide (10%) 30 100.0±0.0 0.0±0.0 Sodium hypochlorite (5-15%) 10 100.0±0.0 100.0±0.0 Sodium hypochlorite (5-15%) 20 100.0±0.0 60.67±0.21 Sodium hypochlorite (5-15%) 30 100.0±0.0 0.0±0.0 Sodium hypochlorite (2,5-7,5%) 10 100.0±0.0 0.0±0.0 Sodium hypochlorite (2,5-7,5%) 20 100.0±0.0 0.0±0.0 Sodium hypochlorite (2,5-7,5%) 30 100.0±0.0 0.0±0.0 Chloramine B (5%) 10 100.0±0.0 20.33±0.11 Chloramine B (5%) 20 100.0±0.0 60.67±0.21 Chloramine B (5%) 30 100.0±0.0 60.67±0.21 Chloramine B (10%) 10 100.0±0.0 100.0±0.0 Chloramine B (10%) 20 100.0±0.0 60.67±0.21 Chloramine B (10%) 30 100.0±0.0 41.0±0.2 Silver nitrate (0,1%) 10 100.0±0.0 80.33±0.11 Silver nitrate (0,1%) 20 100.0±0.0 41.0±0.2 Silver nitrate (0,1%) 30 100.0±0.0 14.67±0.11 Silver nitrate (0,5%) 10 100.0±0.0 0.0±0.0 Silver nitrate (0,5%) 20 100.0±0.0 0.0±0.0 Silver nitrate (0,5%) 30 100.0±0.0 0.0±0.0 Control (distilled water) 30 6.0±0.2 0.0±0.0 4 BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

TABLE 3. Cuitivation of H. officinalis on different types of culture media in invitro Medium Plant characteristics Week 1 of cultivation Week 2 of cultivation Week 3 of cultivation MS1 SL – 2,4 cm; NL –2; DR is not visible; CP – light brown H. officinalis on MS1 medium SL –2,4 cm; NL –2; DR is not visible; CP – intense brown H. officinalis on MS1 medium SL – 2,4 cm; NL –2; DR is not visible; CP – brown; the plant died H. officinalis on MS1 medium MS2 SL – 1,5 cm; NL –4; DR is visible; CP – intense green H. officinalis on MS2 medium SL – 1,9 cm; NL –4; darkening and redness of the root can be seen; CP – green H. officinalis on MS2 medium SL – 3,6 cm; NL –6; DR is visible; CP – green-brown H. officinalis on MS2 medium MS3 SL – 1,8 cm; NL–4; DR is not visible ; CP – intense green H. officinalis on MS3 medium SL – 2 cm; NL –4; DR is not visible; CP – intense green H. officinalis on MS3 medium SL – 2,7 cm; NL–6; DR is visible; CP – intense green H. officinalis on MS3 medium MS4 SL – 0,7 cm; NL–2; DR is not visible; CP – green H. officinalis on MS4 medium SL – 1,2 cm; NL–3; DR is not visible; CP – green with darkening H. officinalis on MS4 medium SL – 4,5 cm; NL –6; DR is not visible; CP – green; callus is formed. H. officinalis on MS4 medium MS5 SL – 1,1 cm; NL –4; DR is not visible; CP – green H. officinalis on MS5 medium SL – 2,2 сm; NL –4; DR is not visible; CP – green H. officinalis on MS5 medium SL – 2,3 cm; NL–9; DR is visible; CP – intense green H. officinalis on MS5 medium 5 BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

4 Сonclusion During the study, the introduction of H. officinalis plant explants into the in vitro culture was carried out, the sterilization process of H. officinalis plant explants was optimized, somel of the most effective seed sterilization modes, their exposure time and concentration were found. The most optimal are 5-15% solution of sodium hypochlorite and 10% solution of chloramine B with its effect for 10 minutes, providing 100% sterilization of plant explants and most importantly 100% germination of viable seedlings. Eight culture media were tested for the cultivation of H. officinalis seedlings. It was found that the most optimal nutrient medium for the induction of callusogenesis was a medium with indolyl-3-butyric acid - 1 mg / l, and a medium with a-naphthylacetic acid - 0.5 mg / l, kinetin - 0.1 mg / l and 6 -benzylaminopurine - 1 mg / l, and for the cultivation of plants H. officinalis in vitro - a medium containing indolyl-3-acetic acid IAA (2 mg / l), and kinetin (0.2 mg / l). Thus, callus tissues and mini-plants of H. officinalis were obtained in vitro and the most optimal nutrient media were determined both for microclonal propagation and for the induction of callus tissue H. officinalis, which can be further used for mass cultivation of cell and culture and production from it safe bioadditives with active substances for livestock and crop production as part of the development of modern agrobiotechnology. Acknowledge This work was carried out as part of a state task FZWG-2020-0021. References 1 S. V. Sizonenko and T. A. Belova, “Hyssopus officinalis to the culture in Medicinal plant growing: from the experience of the past to the latest technologies: materials of the International scientific and practical Internet conference, Poltava, 2012. 2 V. E. Torikov, “Medicinal value of vegetable, fruit and berry, field plants and wild plants,” (Publishing house of the Bryansk state agricultural Academy, Bryansk, 2013) 3 K. S. Timchuk, T. G. Zheleznyak, Z. N. Vorniku, and I. P. Dragalin, “Medicinal Hyssop - the content of essential oil and its component composition,” Materials of the international scientific conference (may 29-31, 2013, Minsk) 4 O. A. Grebennikova, A. E. Paliy, L. A. Khlypenko, V. D. Rabotyagov, Journal "Orbital" 1, 1 (2017) 5 F. Fathiazad, S. Hamedeyazdan, Afr. J. Pharm. Pharmacol 5(17), 1959 (2011) 6 G. Zawiślak, Mod. Phytomorphol. 4, 93 (2013) 7 A. Srivastava, K. Awasthi, B. Kumar, A. Misra, S. Srivastava, India Pharmacogn J. 10(4), 690 (2018) 8 L. A. Kotyuk, Biologichni Studii 7(2), 83 (2013) MS6 SL – 0,6 cm; NL –2; DR is not visible; CP – yellow-brown H. officinalis on MS6 medium SL – 0,6 cm см; NL – 2; DR is not visible; CP – yellow-brown H. officinalis on MS6 medium SL – 0,6 cm; NL–4; DR is visible; CP – green, callus is formed H. officinalis on MS6 medium MS7 SL – 1,8 cm; NL –2; DR is visible; CP – light-brown - green H. officinalis on MS7 medium SL – 1,8 cm; NL –2; DR is visible ; CP – white-brown H. officinalis on MS7 medium SL – 1,8 cm; NL –2; DR is visible; CP – brown – white-brown; the plant died H. officinalis on MS7 medium MS8 SL – 1,3 cm; NL –2; DR is visible ; CP – intense green H. officinalis on MS8 medium SL – 1,5 cm ; NL –4; DR is visible ; CP – intense green H. officinalis on MS8 medium SL – 1,6 cm; NL –4; DR is visible ; CP – intense green; H. officinalis on MS8 medium 6 BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

9 A. N. Shibko and Yu. Aksenov, Ecosystems, their optimization and protection, 4, 127-133 (2011). 10 A. Wesołowska, D. Jadczak, M. Grzeszczuk, Herba Pol. 56(1), 57 (2010) 11 V. Mitić, S. Đorđević, FU Phys. Chem. Tech. 2(2), 105 (2000) 12 M. Moghtader, J. Hortic. For. 6(1), 1 (2014) 13 H. Soleimani, M. Barzegar, M. A. Sahari, B. H. Naghdi J. of Med. Plants, 10(37), 61 (2011) 14 G. Schulz, E. Stahl, Flavor Fragr. J. 6(1), 69 (1991) 15 N. Dehghanzadeh, S. Ketabchi, A. Alizadeh, Asian J. Exp. Biol. Sci. 3(4), 767 (2012) 16 M. Jankovský, T. Landa, Hort. Sci. (Prague), 29(3), 119 (2002) 17 F. Fathiazad, М. Mazandarani, S. Hamedeyazdan, Adv. Pharm. Bull. 1(2), 63 (2011) 18 M. A. Myadelets, D. V. Domrachev, V. A. Cheremushkina, Chemistry of plant raw materials 1, 111 (2012) 19 A. L. Budantsev and E. E. Lesiovskaya, Wild useful plants of Russia (SPKHFA Publishing house, Saint Petersburg, 2001) 20 D. Tančinová, Z. Mašková, D. Foltinová, J. Štefániková, J. Árvay, Potr. S. J. F. Sci. 12(1), 491 (2018) 21 T. G. Kuchina, I. K. Lebedev, and E. A. Kalashnikova, Study of medicinal hyssop (Hyssopus officinalis L.) in culture in vitro modern technologies in the world scientific space collection of articles of the International scientific and practical conference (September 28, 2017, Ufa). 22 B. Hosseini, M. Alizadeh, A. Hassani, M. Jafari, A. Rahimi, J. Medicinal Plants By-products 2, 187 (2016) 23 E. Zayova, M. Geneva, I. Stancheva, L. Dimitrova, M. Petrova, M. Hristozkova, I. Salamon, Med. Plants, 10(4), 295 (2018) 24 I. Stancheva, M. Geneva, M. Hristozkova, E. Zayova, J. Herbs Spices Med. Plants 25(2), 104 2019 25 A.V. Kilchevsky, Genetic bases of plant breeding: Biotechnology in plant breeding. Cell Engineering. (Nauka, Minsk, 2012). 26 T. Murashige, Physiol Plant 15(3), 473 (1962) 27 V. S. Shevelukha, Agricultural Biotechnology. (Vyshaya Shkola, Moscow, 2008) 28 O. A. Timofeeva and N. I Rumyantseva, Culture of plant cells and tissues (Kazan University, Kazan, 2012) 29 Pifo Hans-Peter. Statistics.( Ed. VNIIA, Moscow, 2011) 7 BIO Web of Conferences 30, 05006 (2021) https://doi.org/10.1051/bioconf/20213005006 ILS 2020

Analysis of 16S rRNA gene variability in soil nitrifying bacteria of the genus Nitrosomonas Anastasia Nechayeva*, Konstantin Boyarshin, Olga Bespalova, Violetta Klyueva, Olesya Makanina, and Irina Batlutskaya Belgorod State National Research University, Belgorog, Russia Abstract. The main goal of the work was to assess variability of 16S rRNA gene sequence within the nitrifying bacterial genus Nitrosomonas to find specific sequences for its detection. To achieve it, we had to find and to assess sequences that are highly conservative on the level of the genus and to find and to assess sequences variable on the level of genus but conserved on the level of species. In the SILVA database of ribosomal RNA sequences, 231 sequences of 16S rRNAs of bacteria of the genus Nitrosomonas were collected, of which were sorted 132 sequences by length from 1400 to 1541 (full-sized gene) nucleotides. We conducted an analysis of the taxon-specificity of sequences conserved at the genus level. More than a hundred full matches were found by the BLAST program in the nr database with other genera of the same and other families. So, in Nitrosomonas 16S rRNA gene are present some highly conservative regions, but they are not genus-specific due to high coincidence with other genera. Wherein, a variable region 994-1041 is highly species-specific for the species N. eutropha. Generally, the sequence of 994-1041 region of Nitrosomonas 16S rRNA genes tends to be clustered, being very close between some species. 1 Introduction Nitrification is an important step in the nitrogen cycle in the nature. The nitrification process as a whole consists in the oxidation of organic nitrogen to nitric acid residue. It is carried out in three phases: in the first phase, ammonia (NH4 + ) is formed under the influence of various bacteria, in the second phase, ammonia is oxidized by bacteria of the genus Nitrosomonas etc. to nitrous acid residue (NO2 - ), and finally, in the third phase, it is oxidized by the Nitrobacter genus etc. to nitric acid residue (NO3 - ) [1]. The general scheme of these transformations has the following form: Nitrifying organisms are chemoautotrophs, and use carbon dioxide as their carbon source for growth. Nitrosomonas europaea, as well as populations of soildwelling ammonia-oxidizing bacteria, have been shown to assimilate the carbon dioxide released by the reaction to form biomass via the Calvin Cycle, and harvest energy by oxidizing ammonia to nitrite [2, 3]. Many of such microorganisms also are capable of oxidizing urea and this feature may explain enhanced growth of ammonia-oxidizing bacteria in the presence of urea in acidic environments [4]. Bacteria of the genus Nitrosomonas play a key role in the conversion of ammonium soil nitrogen to nitrite, providing the use of mineral and organic fertilizers by agricultural plants. This genus contains 12 species. In this study, sequence variability of the 16S rRNA gene was evaluated within the nitrifying bacterial genus Nitrosomonas to find specific sequences for its detection, sequences that are highly conserved at the genus level, and sequences that vary at the genus level but remain conserved at the species level were found and evaluated. Analysis of taxon-specificity of sequences that are kept conserved at the genus level was carried out too. 2 Problem statement The presence of nitrate nitrogen in the zone of the root system is of great importance for plants. The nitrifying ability of the soil microbiota reflects its potential in the accumulation of nitrogen usable for plants. In soils with an increased intensity of nitrification processes, a significant amount of nitrates accumulates, thereby creating conditions favorable for denitrification as well [5]. Knowledge of the dynamics of the growth of microorganisms and the intensity of the passage of ammonification and nitrification will allow us to regulate these processes and establish the doses, dates and types of nitrogen fertilizers depending on the soil conditions. *Corresponding author: [email protected] © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 05007 (2021) https://doi.org/10.1051/bioconf/20213005007 ILS 2020

This determines the use of the introduced fertilizers and the reduction of losses during their application. 3 Research question The main question of this study is what DNA sequences can serve as molecular markers to detect the abandons of microorganisms of genus Nitrosomonas in soil microbiota. On this path, we sought to evaluate the sequence variability of the 16S rRNA gene within the nitrifying bacterial genus Nitrosomonas in order to find specific sequences for its detection. To achieve this goal, it was necessary to select a set of sequences that might serve for binding of PCR primers specific for this genus. These should be some evolutionary conservative sequences that are the same for the main representatives of the Nitrosomonas genus. We also needed to find and evaluate sequences that vary at the genus level, but persist at the species level. 4 Purpose of the study The aim of this study was to assess variability of 16S rRNA gene sequence within the nitrifying bacterial genus Nitrosomonas to find specific sequences for its detection. 5 Methods and materials In the SILVA database of ribosomal RNA sequences, 231 sequences of 16S rRNA of bacteria of the genus Nitrosomonas were collected. Of these, using the online interface, they were sorted by the length extracting 132 sequences with length from 1,400 to 1,541 (full-sized gene) nucleotides. The obtained data set contained from one to twelve sequences belonging to each of the twelve species of the genus Nitrosomonas, as well as 63 sequences that were identified on the genus level only. 132 sequences of 12 species and unclassified bacteria of the genus Nitrosomonas were aligned by the Clustal Omega program. This way three conservative sequences more than 20 nucleotides in length have been identified. On the table I traditionally distinguished highly conserved sequences of bacterial (E) and archaeal (A) rRNAs are shown (table I, [6]). These sequences should be too conserved to be genus-specific. But they are not the same even within the same genus, as we show, aligning 132 Nitrosomonas – as we have only three of all these sequences which are truly conservative, and only one of them is completely conservative (the second). The conservative sequences found at the level of the genus Nitrosomonas overlap to some extent with the universal conservative sequences traditionally distinguished in special literature [6] (tab. 2). As known, group-specific PCR is PCR for related sequences within the same or between different species using conservative primer-binding sequences. It means that the selection of universal primers for 16S ribosomal genes for amplification of a species-specific part of the gene implies that the sequence of 16S gene is conserved between species, therefore, amplification of the sequence will be performed for all studied species. As usual, several steps are used to construct groupspecific primers, including collecting data on the sequences of the selected gene from several adjacent taxa, aligning the nucleotide sequences and analyzing them, identifying regions with the optimal level of variability, selecting primer sequences, and checking that the sequences match the molecules in database. The most time-consuming steps: comparing the aligned areas of closely related groups aimed at detecting unique group features, and searching for randomly matching sequences. Table 1. The conservative fragments in archael and eubacterial 16s rDNAs [6]. That is why, for a more simplified obtaining of sequences with degenerate positions, we created a special program based on a high-level general-purpose programming language, aimed at increasing developer productivity and code readability “Python 3.7”. The script package written in “Python 3.7” is developed to facilitate these steps. It includes three main tools. Two of them are destined to build consensus sequences on the base of alignments generated by wellknown “Clustal Omega” tool [7]. Unlike other common programs for building consensus sequences the scripts of the package can insert in them characters marking variable positions and indicating nucleotides that can occupy them. Also, it is possible to represent consensus in form of six lines with percentage of occurrence of every nucleotide or deletion. 2 BIO Web of Conferences 30, 05007 (2021) https://doi.org/10.1051/bioconf/20213005007 ILS 2020

To facilitate analysis of data obtained by “Nucleotide BLAST” package, the tool for comparison of two files containing results of searches in nucleotide sequence database with “BLAST” was developed. It allows fast check whether two primers in the designed pair have coinciding sequences in the same molecule that may lead to false positive results. This greatly simplifies the analysis of possible problems with the specificity of the selected primer pair. In general, the use of the described scripts demonstrates their positive role in solving problems related to the selection of species-specific and group-specific primers. 6 Results As a result, three regions with a length of more than twenty nucleotides were identified, identical for 95 percent or more of the representatives of the genus (Table II). A comparison of the observed sites conservative at the genus level with a map of rRNA sites conservative at high taxonomic levels showed their incomplete overlap. Conservative plot 1169-1255 to the least extent overlaps with the traditionally allocated highly conserved sequences (28% overlap). We also performed an analysis of the taxonspecificity of sequences stored at the genus level. More than a hundred full matches were found by the BLAST program in the nr database with other genera of the same and other families. The conserved sequences found were not specific for the genus. Highly variable sequences are located in regions 1- 104, 994-1041 and 1431-1541. For further analysis, we chose the short sequence 994-1041 in the middle of the molecule. To evaluate the intraspecific and interspecific variability of this region with the help of a computer program, we created sequences from sets of sequences for generating consensus. After making sure that 16S rRNA sequences that are highly conserved at the genus level are not genus-specific, we shifted our attention to highly variable sequences and traced their variability at the species level. Table 2. Partial overlay of the sequences in 16S rRNA gene, conserved on the genus level, with known highly conservative sequences (from table 1). Nitrosomonas species are divided to some clusters by 994-1041 sequence. The alignment of all these twelve consensus sequences depicts high variability (Fig. 1). Based on these data, a tree of consensus sequences was built (Fig. 2). As it turned out, the sequence 994-1041 is highly conservative at the species level, for example, for Nitrosomonas eutropha (Fig. 3). After aligning the consensus sequences, we got the general one with the help of our special program based on a high-level general-purpose programming language, aimed at increasing developer productivity and code readability “Python 3.7”. Then we decided to check on the basis of nr “BLAST” database whether the created consensus sequence would be detected within the species Nitrosomonas eutropha. Fig. 2. Phylogenetic tree of consensus sequences of positions 994-1041 of 16S rRNA gene, built by Clustal Omega program [7]. Fig. 1. Alignment of consensus sequences of positions 994-1041 of 16S rRNA gene of all twelve Nitrosomonas species, built by Clustal Omega program [7]. 3 BIO Web of Conferences 30, 05007 (2021) https://doi.org/10.1051/bioconf/20213005007 ILS 2020

As a result, only three genera, Azoarcus, Thauera and Burgholderia have the same sequence in this region. So, this region is highly specific for N. eutropha and is promising for elaboration of species-specific PCR primers. 7 Conclusion As the result of the work, highly conservative regions of Nitrosomonas 16S rRNA gene are present, but are not genus-specific due to high coincidence with other genera. Conservative sequence sets are often used to generate phylogenetic trees, since it can be assumed that organisms with similar sequences are closely related [3]. The choice of sequences may vary depending on the taxonomic scope of the study [8, 9]. The most highly conserved genes such as 16S ribosomal RNA and other sequences are useful for reconstructing deep phylogenetic relationships and identifying bacterial phyla in metagenomic studies [6]. As is known, the genome of most species of the genus Nitrosomonas has not yet been determined [10]. But fortunately, we were able to determine that sequence of 994-1041 region of Nitrosomonas 16S rRNA tends to be clustered, being very close for some species. Variable region 994-1041 is highly species-specific at least for species Nitrosomonas eutropha. Though the genome Nitrosomonas eutropha has not been determined for all strains, the genome of Nitrosomonas eutropha C91 C71 has been sequenced and investigated extensively [9, 11]. Although the genomes of some Nitrosomonas species have yet to be sequenced, there are many research areas that have benefited from sequencing the more studied representatives of the Nitrosomonas species N. europaea and N. eutropha, including wastewater treatment, agriculture and biogeochemistry. References 1. F. Beeckman, H. Motte, T. Beeckman, Curr. Opin. Biotechnol. 50, 166 (2018) 2. R. Hatzenpichler, Appl Environ Microbiol 78, 7501 (2012) 3. U. Purkhold, A. Pommerening-Roser, S. Juretschko [et al.], Appl Environ Microbiol 66, 5368 (2000) 4. K. L. Marsh, G. K. Sims, R. L. Mulvaney, Biol. Fert. Soil 42, 137 (2005) 5. K. Kazeev, G. Mokrikov, U. Akimenko, M. Myasnikova, S. Kolesnikov, Environmental Assessment of the Use of No-Till Technology in the Rostov Region, Monograph, 2018. 6. Y. Wang, P.-Y. Qian, PLoS ONE 4, e7401 (2009) 7. J. D. Thompson, T. J. Gibson, F. Plewniak, F. Jeanmougin, D. G. Higgins, Nucleic Acids Res 25(24), 4876 (1997) 8. S. R. Gadagkar, M. S. Rosenberg, S. Kumar, J. Exp. Zool. Part B 304B, 64 (2005) 9. L.Y. Stein, D.J. Arp, P.M. Berube [et al.], Environ. Microbiol. 12, 2993 (2007) 10. P. Chain, J. Lamerdin, F. Larimer, W. Regala [et al.], J. Bacteriol 185, 2759 (2003) 11. H.-P. Koops, B. Böttcher, U. Möller, A. Pommerening-Röser, G. Stehr, J Gen Microbiol, 13, 1689(1991) Fig. 3. Alignment of fifteen sequences of region 994-1041 of N. eutropha 16S rRNA gene 4 BIO Web of Conferences 30, 05007 (2021) https://doi.org/10.1051/bioconf/20213005007 ILS 2020

Low homology between 2019-nCoV Orf8 protein and its SARSCoV counterparts questions their identical function Viktoria Iatsenko*, Konstantin Boyarshin, Olga Bespalova, Violetta Klyueva, Yuliya Kurkina, and Irina Batlutskaya Belgorod State National Research University, Belgorog, Russia Abstract. SARS-CoV accessory protein Orf8b is involved in suppressing interferon-mediated immune response of the infected cell and this might lead to supposition that the corresponding protein 2019-nCoV Orf8 shares the same role. But the tertiary structures of these proteins are still unknown, and the primary structures demonstrate very low homology and different calculating parameters. This time they both are affected by stabilizing selection and in natural viral populations do not tend to be deleted. The question whether in this case very different proteins could share the same function rises from the present data. 1 Introduction Single-stranded positive-sense RNA viruses of the family Coronaviridae are capable to infect amphibians, birds and mammals [1]. In particular, they cause such infectious human diseases as Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) [2-4] and CoVID-19 [5]. Viruses close to 2019- nCoV also infect pangolin Manis javanica [6-8] and bats, particularly Rhinolophus affinis [9,10]. Several other human infecting coronaviruses, such as HCoVNL63, HCoV229E, HCoV-OC43 and HCoVHKU1 are well known [11]. These viruses are quite different, so 2019-nCoV and SARS-Cov share only near 70% genomic identity [12]. The closest animal counterpart of 2019-nCoV seems to be the bat virus RaTG13 with 96% genomic identity [10]. So-called accessory proteins of coronaviruses in many cases are involved in blocking of the interferonmediated immune response of the infected cell [13]. Among them polymorphic protein Orf8 is present in 2019-nCoV and SARS-CoV. In the CoViD-19 pathogen and early strains of SARS coronavirus it exists in the form of a single 121-122 aa protein. In many other SARS strains it is splitted for two different proteins Orf8a and Orf8b. They still are coded by one mRNA, and their coding sequences partially overlap (Fig. 1). On some different models it was shown that the SARS proteins Orf8b and joint Orf8ab are involved in blocking of interferon induction [15]. They interact directly with interferon regulatory factor 3 (IRF3) [16] leading to its ubiquitination and degradation, that prevents the induction of interferon synthesis by its activated form. Orf8a this time prevents the fast degradation of Orf8b. Besides in one of models it some way prevents expression of Orf8b [15], that itself is difficult to interpret. Fig. 1. Open reading frames for the joint protein Orf8ab (or simply Orf8) in animal and early human SARS-CoV and splitted Orf 8a and Orf8b in the late human-adapted strains. The nucleotide sequence absent in the strains with splitted Orf8 is marked by arrows. Figure is from the work of Keng and Tan [14]. 2019-nCoV resembles upper variant, but lacks Orf9b and possess Orf10 right of gene N. It seems that comparing the primary sequences of the coronaviral proteins similar to Orf8b we should achieve high homology and the presence of some invariant structural features, ensuring its function that is really important and supported by stabilizing selection. Otherwise, we should see an example of very fast evolution, changing whether function or the structural base of its performing. 2 Materials and methods Sequences of the proteins Orf8 and Orf8a are from the public database of National Institute of Health of USA. Information about genomic polymorphisms of 2019- nCoV is from the database described in the work of von Dorp and coauthors [17]. For multiple sequence alignment the program Clustal Omega was used [18]. The program Protein BLAST [19] was used for search and pairwise alignment of similar sequences by the chosen query. The standard settings were applied. *Corresponding author: [email protected] © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 05008 (2021) https://doi.org/10.1051/bioconf/20213005008 ILS 2020

To assess amino acid composition and determine theoretical values of computational protein parameters, for instance isoelectric point and grand average of hydropathicity, the program ProtParam [20] was applied. 3 Results and discussion Among 11 Orf8b protein sequences belonging to SARSCoV isolated from humans only 2 polymorphic positions were detected. They both are close to C-terminus. That are Lys81Asn and Thr83Ile substitutions. They tend to coincide and are the minority. In position 81 both residues are positively charged, although to varying degrees, in position 83 properties of residue change. Other positions seem to be monomorphic in such a sampling. 14 sequences of the joint SARS-CoV Orf8 protein all were found to be identical. This sample obviously does not exhibit all the variation abilities, but it lets achieve the sequence to be relatively conserved in human samples. Joint Orf8 sequences of SARS-like bat coronaviruses are divided to three completely different types. The most numerous cluster is including 10 closely homologous sequences possessed 17 polymorphic positions, 11 of them in the N-terminal region, if to call so 84 N-terminal residues basing on the length of presumably homologous Orf8b. Aligning 13 2019-nCoV Orf8 sequences, 2 close bat and 2 pangolin ones, 15 polymorphic positions were found in 84 aa arbitrary N-terminal region, besides 16 aa deletion in one of the pangolin sequences. These four groups of protein sequences, aligned and roughly evaluated in terms of their variability, were aligned to visualize common features of their regions presumably corresponding to SARS-CoV Orf8b. The most characteristic sequences were selected to show them, giving an idea of the overall picture (fig. 2). Thus, sequences of joint SARS-CoV and SARS-CoV like Orf8 from samples taken from human (hS), civet (cS) and bat (bS), of human SARS Orf8b (8b), of 2019- nCoV and nCoV-like viruses Orf8 from human (hC), bat (bC) and pangolin (pC) were included in the multiple alignment. SARS-CoV Orf8b has strong homology with some of SARS-like viruses from bats, weak homology with Orf8 from 2019-nCoV and related coronaviruses, and very little similarity to the joint Orf8 (Orf8ab) from some SARS-CoV forms from human and their close relatives from bat and civet. With SARS-CoV with joint Orf8 it shares essentially different positions then with 2019- nCoV. The proteins in our sampling share universally only two amino acid positions: Cys40 (Cys83 by numeration of 2019-nCoV) and Leu56 (Leu98). Such a way, Orf8 protein from SARS pathogens are absolutely divergent not only by splitting of Orf8 coding sequence but also by the history of this sequence. It may be closer to 2019-nCoV in the splitted variant and very far from it in the joint one. However, in both cases the similarities do not seem completely irregular. Percentage of identity between Orf8b and parts of SARS-CoV and 2019-nCoV Orf8 aligned with it is only 17% for SARS fused form and 25% for 2019-nCoV protein (tab. 1). The most impressing is completely different electric charge of both types SARS-CoV and 2019-nCoV molecules, that mirrors isoelectric point (pI) value. The indexes related to hydrophobicity also differ strongly. These data do not testify in favor of functional homology between SARS-CoV and 2019-nCoV Orf8 proteins, even to a greater extent than low levels of identity. It is known that the capabilities of the joint Orf8 and the splitted proteins to bind other viral proteins are different [21]. Keng and Tan deduce from these data some significant conformational rearrangement that occurred because of transition from single to splitted protein [14]. But then only the data about interactions between SARS-CoV proteins were used. Adopting the version of functional homology between 2019-nCoV Orf8 and its SARS-CoV counterparts, we should turn attention to the conclusions of Wong and coauthors, that postulate direct interaction between SARS-CoV Orf8b and Orf8ab with human IRF3 [15]. So, we might expect that their counterpart in 2019-nCoV Orf8 shares this function, but too different protein parameters question this point. The obtained data do not claim to be the final solution to this question, but they draw attention to significant differences among coronaviruses designated as SARS-CoV, as well as to the deep divergence of Orf8 proteins. In this regard, attention should be paid to the independent adaptation of SARS-CoV and 2019-nCoV to reproduction in human cells. In the case of commonality of their functions, we have an example of convergent evolution, which went significantly different ways. If the functions have changed, experimental studies can help clarify this complex picture. The expectation of structural information about the considered proteins becomes more intriguing in the presented context. Table 1. Comparision of identity percentage between SARSCOV ORF8B (8B), another SARS-COV joint ORF8 (HS, designation as on alignment on fig. 2) and 2019-NCOV ORF8 (HC), and some calculated parameters of these sequences. Length: full (identity), homologous part (identity) pI Aliphatic index GRAVY 8b 84 9.45 88.21 -0.029 hS 122 (11%) 8.24 96.72 0.218 84 (17%) 8.55 88.33 0.038 hC 121 (17%) 5.42 97.36 0.219 81 (25%) 5.18 99.75 0.143 . 2 BIO Web of Conferences 30, 05008 (2021) https://doi.org/10.1051/bioconf/20213005008 ILS 2020

XXX-X-XXXX-XXXX-X/XX/$XX.00 ©20XX IEEE Fig. 2. Aligned sequences of joint SARS-CoV and SARS-CoV like Orf8 from samples taken from human (hS), civet (cS) and bat (bS), of human SARS Orf8b (8b), of 2019-nCoV and nCoV-like viruses Orf8 from human (hC), bat (bC) and pangolin (pC). Amino acid residues matching with those of SARS-CoV Orf8b (8b) are marked gray. References 1. L.P. Shaw, A.D. Wang, D. Dylus [et al.], Mol. Ecol. 29, 1 (2020) 2. Z. Song, Y. Xu, L. Bao, L. Zhang [et al.], “Thrusting Coronaviruses into the Spotlight”, From SARS to MERS Viruses, 11 (2019). 3. Y. Yin, R.G. Wunderink, Respirology 23, 130 (2018) 4. E. de Wit, N van Doremalen, D Falzarano, V.J. Munster, Nat Rev Microbiol. 14, 523 (2016) 5. C. Huang, Y. Wang, X. Li [et al.], Lancet 395, 497 (2020.) 6. K. Xiao, J. Zhai, Y. Feng, [et al.], Nature onlinepublication (2020) 7. L. Wahba, N. Jain, A. Fire [et al.], bioRxiv onlinepublication (2020) 8. T. Lam, M. Shum, H.-H. Zhu, [et al.], Nature 583, 282 (2020) 9. W. Ji, W. Wang, X. Zhao, J. Zai, X. Li, J. Med. Virol. 92, 433 (2020) 10. P. Zhou, X.-L. Yang, X.-G. Wang [et al.], Nature, 579, 270 (2020) 11. Z. Song, Y. Xu, L. Bao [et al.], Viruses 11, (2019) 12. Y. Z. Zhang, E. C. Holmes, Cell 181, 223 (2020) 13. J. Cui, F. Li, Z.L. Shi, Nat. Rev. Microbiol. 17, 181 (2019) 14. CT. Keng, YJ. Tan, Berlin, Heidelberg, 177-191, (2009) 15. H.H. Wong, T.S. Fung, Sh. Fang [et al.], Virology 515, 165 (2018) 16. K.A. Fitzgerald, S.M. McWhirter, K.L. Faia, [et al.], Nat. Immunol. 4, 491 (2003) 17. L. van Dorp, M. Acman, D. Richard [et al.], Infect. Genet. Evol. 83, (2020) 18. F. Sievers, A. Wilm, D. Dineen [et al.], Mol. Syst. Biol. 7, (2011) 19. S. Altschul, W. Gish, W. Miller, E. Myers, D. Lipman, J. Mol. Biol. 215, 403 (1990) 20. E. Gasteiger, C. Hoogland, A. Gattiker, S. Duvaud, M.R. Wilkins, R.D. Appel, A. Bairoch, Protein Identification and Analysis Tools on the ExPASy Server, ( John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press, 2005) 21. C.T. Keng, Y.W. Choi, M.R. Welkers, [et al.], Virology 354, 132 (2006) 3 BIO Web of Conferences 30, 05008 (2021) https://doi.org/10.1051/bioconf/20213005008 ILS 2020

Effect of liquid organic fertilizer “Efflurost” on economically valuable indicators of vegetable crops Nadejda Kotsareva1* , Petr Tolmachev2 , Vladimir Bredihin3 , and Dmitrii Ohrimchuk4 1FSBEI HE Belgorod State Agricultural University named after V.Gorin, Belgorod, Russia 2LLC “AGROSNABSPAVKA”, Belgorod, Russia 3 LLC “AltEnergo”, Belgorod, Russia 4 LLC “Belgorod Institute of Alternative Energy”, Belgorod, Russia Abstract. The results of determining the application dose of liquid organic fertilizer “Efflurost” and its effect on economically valuable indicators of sweet pepper and eggplant, agrochemical parameters of the soil are presented. The positive effect of liquid organic fertilizer “Efflurost” on economically valuable indicators of vegetable crops (sweet pepper and eggplant) and on the soil agrochemical parameters has been established. The optimal application rates of liquid organic fertilizer “Efflurost” for sweet pepper is 1:10 dilution and 1: 5 for eggplant, which bring to yield enhancement of marketable products by 10.6 t / ha and 27.2 t / ha respectively. Soil acidity decreasing by 0.1-0.4 units, the number of microorganisms involved in the mineralization of humic substances and increasing the number of ammonifying microorganisms were noted. 1 Introduction For many years the Belgorod region has been a leader in the meat production. Today the Belgorod region ranks second in pork production among the federation subjects and on the top in poultry production. The volume of meat and poultry production in the region is more than 1.7 million tons per year. Along with the positive aspects the industrialization of livestock breeding brings certain problems - utilization of a huge amount of agricultural waste (manure and bird droppings, processing livestock and crop products waste) with the future use as organic fertilizers [1]. Under scientific approach the efficient use of agricultural waste allows us to reduce mineral fertilizers purchase significantly and to produce own organic fertilizers. According to the current "Rules for the disinfection of manure, litter and sewage" as well as the "Veterinary and sanitary rules for preparing manure and sewage for use as organic fertilizers under infectious and invasive diseases of animals and birds" one of the methods of disinfection and preparation for use of organic fertilizers is their processing in anaerobic conditions. During anaerobic treatment biomass undergoes biodecontamination as a result of which nonspore-forming pathogens of infectious diseases, viable larvae and worm eggs, flies’ pupae and larvae, intestinal protozoa cysts die [2, 3, 4]. Biogas station "Luchki" of "AltEnergo" company produces three types of organic fertilizers. In anaerobic mesophilic reactors biomass (pork and cattle runoff, * Corresponding author: [email protected] livestock and crop production waste) under the influence of hydrolysis reactions and various groups of microorganisms at the temperature of 38-41 °С for 30-50 days is processed into biogas and three types of organic fertilizers, one of which is liquid organic fertilizer “Efflurost”. 2 Experimental The research of the effect of liquid organic fertilizer “Efflurost” on economically valuable indicators of vegetable crops (sweet pepper and eggplant) was carried out at the Department of Horticulture, Selection and Vegetable Production at the Belgorod State Agricultural University named after V. Gorin in 2019. To achieve these goals, the following tasks were set and solved: phenological and biometric measurements were carried out on sweet pepper and eggplant, leaf diagnostics and soil agrochemical analysis were done, the optimal doses of liquid organic fertilizer "Efflurost" for sweet pepper and eggplant were determined. The laying of the experimental plots and the processing of the obtained data were carried out according to existing methods [5, 6]. Liquid organic fertilizer "Efflurost" was added under pepper and eggplant - 1 l / per 1 plant with a dilution of 1: 3, 1: 5, 1:10, the control is water. The sample of pepper and eggplant is 20 plants for each option. Phenological observations and plants biometric measurements were done before application of liquid organic fertilizer "Efflurost" and in 10 days. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 05009 (2021) https://doi.org/10.1051/bioconf/20213005009 ILS 2020

Soil analysis can`t demonstrate how efficiently plants absorb nutrients from the soil. Plant tissues analysis is a fairly economical and strategic tool for monitoring of plants growth and development; therefore, it was done leaf diagnosis and soil agrochemical analysis according to the options. Leaf diagnosis allows us to determine the condition of plants during blossom time as well as nutrition problems at the moment. Leaf diagnosis is especially important for adverse external factors that make it difficult for the root system to absorb nutrients from the soil. Leaf diagnosis can demonstrate the current plants state and in particular a deficit of one or another element. Leaf diagnosis used during blossom time can be visual, chemical and functional. 3 Results and discussions The effect of liquid organic fertilizer “Efflurost” on the increasing of crops vegetative mass was multivalued. The largest increase in tops for 10 days after application of liquid organic fertilizer “Efflurost” was observed in sweet pepper plants in the dilution of 1:10 (Fig.1). Plant gain of sweet pepper in the control amounted to 14% and with a 1:10 dilution of liquid organic fertilizer “Efflurost” the height increased by 81%. The best results of plant gain of eggplant were obtained at the dilution 1: 3 and 1: 5 (84% and 93%) (Fig.2). During leaf diagnosis of sweet pepper in the control and at a dilution of 1: 3 it was determined overdose of nitrogen - 11%, while at a dilution of 1: 5 and 1:10 it was found a lack of nitrogen - 10%. A lack of phosphorus and potassium has been also noted. During leaf diagnosis of eggplant it was determined overdose of nitrogen in the control - 15.5%, in the option with a dilution of 1: 3 - 10%, with a dilution of 1:10 - 14%. A lack of phosphorus was found in the control and with a dilution of 1: 3. Overdose of potassium was found in eggplant leaves at a dilution of 1: 3 - 16.3%, a lack of potassium was found at a dilution of 1:10 of liquid organic fertilizer “Efflurost”- 19.6%. The effect of application of liquid organic fertilizer “Efflurost” on the yield indicators of vegetable crops was determined (table 1). The mass of sweet pepper fruits varied from 37.5 g in the control to 39.0 g according to the application options. There were from 13 fruits in the control to 16-18 ones in the options. The largest yield gain was noted in sweet pepper when using liquid organic fertilizer “Efflurost” at a dilution 1:10, which was 10.6 t / ha. The fruit weight of eggplant was 194-195 g with a load of 9-11 fruits per plant. The gain from the application of liquid organic fertilizer “Efflurost” was greatest at a dilution of 1: 5 –27.2 t / ha. The soil agrochemical analysis was done at the end of growth. As a result of the soil agrochemical analysis it was noticed decrease of soil acidity by 0.1-0.4 units under application of liquid organic fertilizer “Efflurost” (Table 2). Fig. 1. Plant gain of sweet pepper depending on the concentration of liquid organic fertilizer “Efflurost”. Fig. 2. Plant gain of eggplant depending on the concentration of liquid organic fertilizer “Efflurost”. The mass content of total nitrogen and phosphorus in the soil decreased under application of liquid organic fertilizer “Efflurost” at a dilution of 1:3 and 1:10 and the potassium indicators increased in comparison with the control. Beneficial microorganisms contribute to the conversion of insoluble soil elements and fertilizers into accessible forms, fix atmospheric nitrogen and release biologically active substances: vitamins, growth stimulants and other useful substances. At the same time, they can also cause negative processes for plants: biological immobilization, gaseous nitrogen decline during denitrification and some microbes release poisons. Under application of liquid organic fertilizer “Efflurost” at a dilution of 1:3 it was noticed decreasing of the number of ammonifying microorganisms up to 5.3x106 CFU/g, but the number of microorganisms using mineral forms of nitrogen decreased from 4.8x106 CFU/g in the control to 4.7x106 CFU/g at a dilution of 1: 3 and up to 4.5x106 CFU/g at a dilution of 1:10. It was noted a decreasing of microorganisms involved in the mineralization of humic substances from 3.7x106 CFU/g in the control to 3.0x106 CFU/g. 2 BIO Web of Conferences 30, 05009 (2021) https://doi.org/10.1051/bioconf/20213005009 ILS 2020

Table 1. Effect of liquid organic fertilizer “Efflurost” on the yield of vegetable crops. Crop Options Mass, g Fruits per plant, units Crop yield, t/ha ± to the control Sweet pepper Water is the control 37,5 13 26,7 - 1:3 38,5 16 32,6 +5,9 1:5 38,5 16 34,9 +8,2 1:10 39,0 18 37,3 +10,6 least mean difference 05 5,2 Eggplant Water - control 194 9 52,4 - 1:3 195 10 74,2 +21,8 1:5 197 11 79,6 +27,2 1:10 195 9 67,3 +14,9 least mean difference05 9,5 Table 2. Soil agrochemical analysis under application of liquid organic fertilizer “Efflurost”. 4 Conclusion The positive effect of liquid organic fertilizer “Efflurost” on economically valuable indicators of vegetable crops (sweet pepper and eggplant) and on the soil agrochemical parameters has been found. The optimal application rates of liquid organic fertilizer “Efflurost” have been determined to yield enhancement of vegetable crops and soil fertility. The largest yield gain was noted in sweet pepper under application of liquid organic fertilizer “Efflurost” at a dilution of 1:10 and in eggplant at a dilution of 1:5. References 1. V.D. Solovichenko, S. I. Tyutyunov, V. V. Nikitin, and E. V. Navoleneva, “Pork runoff is a valuable organic fertilizer”, Scientific-production manual, Belgorod: Publishing House “Otchiy kray", p. 28 (2017) 2. “Instruction for the organic fertilizer production resulting from biomass anaerobic processing”, Belgorod: LLC Altenergo, p.18 (2018) 3. TU 20.15.80-001-76522675-2018. “Organic fertilizer based on pig manure”, URL: // www: // http: //docs.cntd.ru/document/437232067. 4. “Veterinary and sanitary rules for the preparation of manure, litter and sewage for the use as organic fertilizers in infectious and invasive diseases of animals and birds” // URL: // https: //www.fsvps.ru/fsvps/laws/164.htm. 5. B.V. Dospehov, “Methodology of experimental work”( Kolos, Moscow, 1982) 6. S. S. Litvinov, “Methodology of experimental work in vegetable growing (GNU VNIIO, Moscow, 2011) Indicators Application options Without application Dilution 1:3 Dilution 1:10 Salt extract рН, units 6,9±0,1 7,0±0,1 7,3±0,1 Total nitrogen content , % 0,23±0,03 0,22±0,03 0,21±0,03 Total phosphorus content, % 0,22±0,016 0,20±0,014 0,20±0,014 Total potassium content, % 1,88±0,24 1,94±0,24 1,94±0,24 Microbiological indicators (depth 15 sm) Ammonifying microorganisms , CFU/g 7,6х106 7,7х106 7,9х106 Microorganisms using mineral forms of nitrogen (including actinomycetes),CFU/g 3,0х106 3,0х106 2,9х106 Microorganisms involved in humic substances mineralization, CFU/g 1,7х106 1,9х106 1,9х106 Azotobacter (nitrogen-fixing microorganisms), % 60 60 60 3 BIO Web of Conferences 30, 05009 (2021) https://doi.org/10.1051/bioconf/20213005009 ILS 2020

Recent achievements in the field of research of pathogens and methods of treatment and prevention of Mortellaro Disease Natalya Belyakova 1* , Anatolyi Kovalenko1 , Yuliya Bodrova1 , and Viktoriya Oskolskaya1 1 Department of Infectious and Invasive Pathology Belgorod State Agricultural Academy, vil.. Mayskiy, Russia Abstract. In the course of research on the search for substances active in the fight against Mortellaro’s disease, a drug for the treatment and prevention of skin and skin diseases was developed and tested on the basis of zinc and copper metals immersed in the niosomal structures with the addition of Dimethicone copolyol. These components were selected as proven antiseptic. The therapeutic effect was compared at different concentrations of metals in the drug. Practical experiments have shown a high therapeutic effectiveness of the received drug for the treatment of skin and skin diseases of animals, including Mortellaro disease. 1 Introduction Mortellaro disease is an infectious disease of the hooves of cattle, affecting mainly the hind limbs, transmitted through the environment, the causative agents of the disease is a combination of bacteria. [1] The classification of lesions, by which the severity of the process of Mortellaro disease is standardly determined, was developed by Döpfer and expanded by Berry [2]. Classification describes six stages of the disease: M0- animal healthy, M1-active granulomatous area 0-2 cm, M2-ulcerative lesion with an area of more than 2 cm, M3- ulcerative lesion covered with a scab, M4-skin change with signs of Mortellaro disease and M4. 1-recurrent lesion. [3] Over the past decade, studies of pathogens of Mortellaro disease have revealed a wide range of organisms – potential agents of disease development. Researchers in most countries have concluded that the predominant morphotypes detected from active lesions of the disease and the main pathogens of Mortellaro disease are treponemes, which differ in phylogenetic and serological characteristics [4-11]. The Resulting Treponema species are defined in three groups: Treponema medium / Treponema vincentii-like, Treponema phagedenis-like, and Treponema denticola/Treponema putidum-like, the latter now classified as a new species, Treponema pedis [12]. The pathogenesis of the disease is associated with the penetration of Treponema into the skin of the hoof after damage to the epidermis and keratin layer. Loss of the keratin layer is the primary sign of the development of Mortellaro disease. It is assumed that Treponema pathogens secrete keratolytic toxin at an early stage of the disease [13]. This is accompanied by hyperplasia and hypertrophy of the epithelium to more than 100 cells per * Corresponding author: [email protected] 1 mm, with a normal thickness of 5 – 70 cells (Blowey et al., 1994; Döpferet al., 1997). [14] Dichelobacter nodusus bacteria, secreting proteolytic enzymes, destroy successively the surface and Central layers of the epidermis, after which spirochetes of the PT1, PT3, PT6, PT8 and Treponema brennaborense phylotypes that live in the deep layers of the epidermis penetrate the unprotected skin [15, 16]. Treponema spp. it can undergo morphological changes from helical to cysteated forms [17] The Transition to the form of cysts occurs inside the host as a manifestation of the protective mechanism and one of the ways of transmitting the infectious agent in the external environment [18]. The transition requires a temperature of 37° Celsius and an anaerobic environment, which is quite achieved in the deep layers of the hoof skin. Based on the data obtained from the study of 12 farms in the Netherlands, scientists I. Demirkan, M. Erdoğan et al. we calculated the reproduction index of Mortellaro disease, which was 2.36. The active stages of the disease were considered Infectious. The transition from the M0 stage in 94% occurs in the M4 stage and the greatest part of the time the disease on farms occurred in the M4 stage, determining 88.5% R0. As dictated by scientists, preventing damage to the M4 would reduce R0 to unity and below. Treatment on farms occurs mainly in the active stages of the disease – M2 and M3, when there is a distinct lameness of the animal. [2] The M4 stage does not respond well to standard prevention in the form of hoof baths and antibiotic therapy due to the deep occurrence of cysts in the layers of the skin and subcutaneous tissue of the animal's hoof [19-21] but it is the elimination of this stage that will lead to a complete recovery of the herd. Therefore, further research on effective prevention and treatment of class M4 is crucial. Medicine of the last decade has been actively searching for controlled carriers for the targeted delivery © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

of medical components to the site of the disease focus. They improve the therapeutic effectiveness of drug molecules by slowing clearance from circulation, protecting the drug from the biological environment and limiting the impact on target cells. This research is taking place in the field of nanotechnology. The most accessible nanocapsules to create and use are liposomal forms. However, the stability of these substances is relative, due to their organic origin. Their analogues-synthetic niosomes, in terms of bioavailability and protective properties, are not inferior to and in many respects surpass their predecessors. Niosomes are composed of lipid bilayer of non-ionic surfactant, optionally containing cholesterol and its derivatives. The size of these vesicles is from 50 to 800 nm. They have the ability to overcome all the immune barriers of the body, including transdermal, and penetrate the necessary organs and tissues. For the treatment of infectious diseases of the skin and skin, which include Mortellaro disease, for many years, with high therapeutic indicators, copper and zinc metal salts are used, which have bactericidal properties and are able to inactivate the pathogens of Mortellaro disease, violating the membrane structures of the microorganism. To achieve higher treatment rates, the metal must be delivered to the pathogenic organism by enclosing it in a niosomal capsule. Based on this principle, we have developed a drug for the treatment of infectious diseases of the skin and skin of the distal extremities (Mortellaro disease) based on niosomes of copper and zinc salts. During the development of a suitable composition, a clinical trial of five samples of the drug with different ratios of niosomes of metal salts was conducted. The results are recorded and analyzed. 2 Experimental Materials and methods. The work was performed based on the Department of infectious and invasive pathology, laboratory of infectious and invasive pathologies and testing of veterinary drugs of the Belgorod state agrarian University named after V. ya. Gorin. Scientific and production experiments were conducted based on the livestock farm of LLC "Green valley" of the Belgorod region. The study of the features of clinical manifestations of skin and skin lesions of the distal extremities in Mortellaro disease was carried out directly in the conditions of a dysfunctional farm for diseases of the distal extremities of cattle. Clinical assessment of the intensity of lesions in Mortellaro disease was carried out according to the classification proposed by Döpfer, both before the beginning of research and during the course of therapeutic measures. Statistical processing of the obtained results was performed using the student's coefficient. Development of a drug for the treatment of diseases of the distal extremities using niosomal forms of zinc oxide and copper sulfate and available ointment base. On the basis of the laboratory of our university, several experimental samples of the product were created, consisting of an ointment base and niosomal forms of copper sulfate and zinc oxide distributed in its volume in concentrations from 0.2 to 2% of metals in solution, the surface of which was specially modified to ensure high efficiency on the skin and keratinized tissues. The size of niosomes reaches 30-80 nanometers. Niosomes are constructed from a shell in the form of a water-insoluble double layer of non-ionic emulsifier (surfactant), which belong to the group of dimethiconopolyols, which are ethers of polyethylene glycol and polydimethylsiloxane base and are enclosed within the capsule of the active substance. Transdermal transport of zinc oxide and copper sulfate occurs due to the ability of a non-ionic surfactant to form a double molecular layer, similar to the plasma membrane of a living cell. The presence of a si-O-Si covalent bond in the hydrophobic part of the polydimethylsiloxane base of the emulsifier, which has a high elasticity and reactivity, allows delivering a wide range of active substances using reactive sites and purposefully releasing them from the nanovesicle. Thus, Dimethicone copolyol represent hybrid silicon (Dimethicone) and carbon (polyethylene glycol). Sample 1 Niosomes of zinc oxide,% Niosomes of copper sulfate, % 0,1 0,1 Sample 2 Niosomes of zinc oxide,% Niosomes of copper sulfate, % 0,15 0,1 Sample 3 Niosomes of zinc oxide,% Niosomes of copper sulfate, % 0,15 0,15 Sample 4 Niosomes of zinc oxide,% Niosomes of copper sulfate, % 0,2 0,2 Sample 5 Niosomes of zinc oxide,% Niosomes of copper sulfate, % 0,8 0,5 Conducting control tests to study the effectiveness of the drug on sick animals. With each experimental sample of the drug, clinical studies were conducted for various forms of the Depfer pathological process, starting from M1, ending with M4, while taking into account the physiological lactation period in each group of cows and the size of the lesion dimension. Analyzing the data in table 1, it should be noted that sample #1 caused an active granulation process in animals with small M1 lesions with a red or white-red surface and possible exudation as early as 6 days after the start of treatment. The process was accompanied by enlightenment of the affected area and the appearance of pale pink epithelium. Sample # 2, used for treatment in the second group of animals, which contained 12 heads, allowed achieving the same result for 11 animals already on day 5. As for samples 3-5, the same regeneration processes were observed in all tested groups of animals in the 2 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

corresponding number of animals, which was on average 96% effective. Analyzing the data in table 2, we note that cows 2 lactation respond to treatment of M1 lesions to a slightly lower degree. In group 1, the percentage of recovered animals was 95%, in group 2 – 94%, and among group 3, the percentage of recovered animals was also 89%. Sample #4 showed 94 % effectiveness, and among 5 groups, recovered was 100%. As the concentration of the drug increased, the response to treatment occurred on day 6 and on day 3, respectively. According to table 3, the overall effectiveness of treatment was as high as in cows of the second lactation; however, based on the results of treatment indicators by day, it is clear that the therapeutic effect was achieved at a later date. For samples 1-3 - on the 6th day of treatment. The appearance of healing processes for samples 4 and 5 occurred already on the 3rd day, which indicates their higher therapeutic effectiveness. For cows of the fourth lactation, the overall recovery rate was lower than for cows of the first, second and third lactation - 94%. In addition, the response to treatment with a low concentration of the drug (1, 2 and 3 samples) was recorded only on the 6th and 7th days of treatment (table 4). However, for samples #4 and 5, the regeneration process also occurred on the 3rd day after the start of treatment. The indicator of therapeutic effectiveness for cows of the fifth lactation is significantly lower-92 %, which indicates a low kurabelnost of age-old cows in General. However, the use of samples 4 and 5 showed that the drug at a concentration of 0.4% of the total concentration of metals in the solution is able to induce regeneration and healing of areas affected by purulent-necrotic ulcers in aged cows for 3 days, which is comparable to the indicators of the treated effectiveness of these samples in cows of the first, second and third lactation. Table 1. Cows of the first lactation with the development of a pathological process corresponding to M1 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 14 + 13 93% 2 12 + 11 92% 3 17 + 16 94% 4 18 + 18 100% 5 14 + 14 100% Subtotal 75 72 96% Table 2. Cows of the second lactation with the development of a pathological process, corresponding to M1 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 21 + 20 95% 2 18 + 17 94% 3 18 + 16 89% 4 18 + 17 94% 5 17 + 17 100% Subtotal 92 87 95% Table 3. Cows of the third lactation with the development of a pathological process corresponding to M1 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 19 + 18 95% 2 15 + 14 93% 3 10 + 10 100% 4 12 + 12 100% 5 16 + 15 94% Subtotal 72 69 96% Table 4. Cows of the fourth lactation with the development of a pathological process corresponding to M1 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 17 + 15 88% 2 17 + 16 94% 3 18 + 17 94% 4 10 + 10 100% 5 12 + 12 100% Subtotal 74 70 94% 3 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

Table 5. Cows of the fifth lactation with the development of a pathological process corresponding to M1 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 8 + 8 100% 2 11 + 10 91% 3 10 + 8 80% 4 8 + 8 100% 5 13 + 12 92% Subtotal 50 46 92% Cows of the first lactation with M2 lesions, called "classic ulcers", which looked like red or white-red surfaces with a diameter of more than 1 cm with granulation or proliferative tissue (in large quantities or filamentously) and are average in severity of the process, respond to treatment fairly quickly relative to the severity of the process (table 6). Even the minimum concentration of the drug gives a positive therapeutic effect for 6 days after the start of treatment. Optimal rates of response to treatment are also shown in samples 4 and 5. Moreover, in this case, sample 4 showed a greater percentage of efficiency than sample 5. Due to the large variation in the effectiveness of images 1-3 and 4-5, the overall effectiveness of treatment was quite low. Analysis of the table 7 showed that cows of the second lactation with hoof lesions characteristic of M2 according to the Depfer classification could be treated similarly to cows of the first lactation. The percentage of recovered animals in the groups increases relative to the increase in concentration of samples of the drug from # 1 to #4 and does not significantly change for the sample #5 with the highest concentration. However, the overall effectiveness of treatment of second-lactation cows has reached 90%. According to the results of the table 8, the average effectiveness of treatment of groups of animals during the third lactation reached 89%. At the same time, the rate of response to treatment decreased, and for samples # 1-3, the initial stage of proliferation and attenuation of inflammation occurred on the 7th day of treatment. Samples with the maximum concentration also showed a high rate of occurrence of the therapeutic effect: on the 4th and 3rd days, respectively. For cows of the fourth lactation, the timing of the response to treatment has shifted even more compared to animals of the first, second and third lactation. Tightening of the borders of lesions, lightening of inflammation occurred by the end of the first week after the use of the drug. Sample # 4 showed 100% therapeutic effectiveness, but the treatment period also shifted to the end of the week. The overall percentage of recovered cows after fourth lactation fell to 86%, from which it can be concluded that cows respond to treatment worse with age (table 9). The total number of cows of the fifth lactation in the farm is less than other lactations (table 10). The effectiveness of treatment of such cows is apparently lower than that of previous lactation cows. This confirms the correlation between age and treatment duration. Samples 4 and 5 with the maximum concentration of active substances in the composition showed 90% effectiveness of treatment with the appearance of visible regenerative manifestations on the 6th and 5th days of treatment, respectively. The percentage of effectiveness of samples # 1-3 was quite low for the treatment of Mortellaro disease of the second M2 stage of the course and did not exceed 73%. Lesions of the hooves of cows with the First stage of Mortellaro disease are characterized by an acute form of flow, the presence of black necrotic scabs of tissue, and bleeding. This stage is considered as the most severe course of the process of Mortellaro disease. Nevertheless, the therapeutic effectiveness of samples of the drug # 4 and # 5 reached 94 and 100%, respectively. Signs of recovery in cows were observed on day 6 for two samples. The efficiency of samples No. 1-3 were also high, ranging from 75 to 80%. The timing of the appearance of signs fell on the 7th day(table 11). Analysis of the table 12 data showed that the developed tool copes with severe skin lesions in Mortellaro disease with an efficiency of 91% with the most concentrated sample and 87% with the use of sample #4 in sick cows of the second lactation. When testing sample # 1 with minimal concentration, recovery processes began to appear only in the second week after the start of treatment. Therefore, for the treatment of severe Mortellaro disease, this sample is not suitable due to the duration of the process. Samples # 2 and #3 showed sufficient efficiency: 81 and 85%, respectively. According to the table 13, treatment of cows of the third lactation with the disease of Mortellaro stage M3 - "acute", showed a high therapeutic effectiveness of the drug in General: 85% of the number of patients. The best performance indicators were achieved with the use of samples #4 and #5 – both showed 94% therapeutic effectiveness at the same time of recovery (on the 6th day). This shows that the concentration of sample #4 is sufficient for the treatment of severe stages of the disease among cows of the third lactation. The effectiveness of samples #4 and #5 in the treatment of cows of the fourth lactation with the First stage of the disease was 92 and 93%, respectively (table 14). These indicators are slightly lower than in the treatment of cows with severe lesions of the third lactation. 4 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

Table 6. Cows of the first lactation with the development of a pathological process corresponding to M2 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 23 + 19 83% 2 24 + 21 87% 3 15 + 13 87% 4 18 + 17 94% 5 22 + 20 91% Subtotal 102 90 88% Table 7. Cows of the second lactation with the development of a pathological process corresponding to M2 lesions according to the dopfer classification. Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 22 + 18 81% 21 + 19 86% 15 + 14 93% 17 + 16 94% 19 + 18 95% 94 85 90% Table 8. Cows of the third lactation with the development of a pathological process corresponding to M2 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 14 + 11 78% 2 12 + 11 92% 3 15 + 14 93% 4 13 + 12 92% 5 14 + 13 93% Subtotal 68 61 89% Table 9. Cows of the fourth lactation with the development f a pathological process corresponding to M2 lesions according to the dopfer classification Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 13 + 9 69% 2 14 + 12 86% 3 12 + 10 83% 4 12 + 12 100% 5 14 + 13 93% Subtotal 65 56 86% There is also a correlation between age and response time to treatment. Indicators of the effectiveness of the treatment may be considered high. The overall therapeutic effectiveness was 83% of the number of patients. According to the table 15, sick cows of the fifth lactation succumbed to treatment slightly worse than cows of the fifth lactation, which was assumed, the therapeutic effectiveness was 83%. The response to treatment occurred on day 6 for samples # 4 and #5. For sample # 3, the therapeutic effectiveness was 87%, and this indicator can be considered high. Indicators of samples #1 and #2 are also considered satisfactory, but the duration of exposure to the drug to achieve the necessary healing effect is up to one and a half weeks or more. Treatment of cows of the first lactation with M4 lesions, which are characterized by skin changes with the use of a proliferative form (table 16). Before applying therapeutic samples, the lesion was a tightening wound surface covered with a crust. This process passes under favorable conditions to the stage of complete recovery or to a chronic relapsing form. Treatment of typical M4 stage lesions involves achieving complete asepsis of the wound surface to eliminate the risk of returning to the toxic effect of pathogenic microflora, which entails rapid regeneration of the affected tissues. The overall therapeutic effectiveness of the treatment was 95% after the use of all 5 samples, including for the sample #1- 87%, #2 – 94%, #3 – 93%. For samples # 4 and # 5, the therapeutic efficacy was 100%. The onset of recovery occurs on the 4th day for samples # 1-3 and on the 3rd day-for # 4 and #5 (table 17). Indicators of therapeutic effectiveness of the drug in samples # 4 and # 5 reached 100%. The overall therapeutic effectiveness of treatment of second lactation cows was 96%. For images #1-3, the indicators were also high (94, 94, and 93%, respectively). Cows of the third lactation with M4 stage lesions responded to treatment as early as 4 days after testing 1-3 samples and 3 days for 4 and 5 samples. The treatment efficiency was 94% and increased for each group according to the sample concentrations (table 18). 5 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

Table 10. Cows of the fifth lactation with the development of a pathological process corresponding to M2 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 11 + 8 73% 2 12 + 9 75% 3 10 + 7 70% 4 9 + 8 89% 5 10 + 9 90% Subtotal 52 41 79% Table 11. Cows of the first lactation with the development of a pathological process corresponding to M3 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 16 + 12 75% 2 19 + 15 80% 3 15 + 13 86% 4 18 + 17 94% 5 15 + 15 100% Subtotal 83 72 86% Table 12. Cows of the second lactation with the development of a pathological process corresponding to M3 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 14 ± 10 71% 2 16 + 13 81% 3 14 + 12 85% 4 15 + 13 87% 5 11 + 10 91% Subtotal 70 58 83% Table 13. Cows of the third lactation with the development of a pathological process corresponding to M3 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 15 ± † 10 67% 2 13 + 11 85% 3 14 + 12 86% 4 16 + 15 94% 5 17 + 16 94% Subtotal 75 64 85% Table 14. Cows of the fourth lactation with the development of a pathological process corresponding to M3 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 13 ± ‡ 9 69% 2 14 ± § 11 76% 3 13 + 11 85% 4 12 + 11 92% 5 16 + 11 93% Subtotal 68 57 83% Cows of the fourth lactation respond to treatment, respectively, cows of the third lactation: 94% and with a response to treatment on the 4th day for 1-3 samples and on the 3rd day for 4 and 5 samples (table 19). For cows of the fifth lactation, the overall therapeutic effectiveness was 94%, but the timing of the reaction to † Incomplete manifestation of healing processes up to 7 days, recovery on the 9th day ‡ Incomplete manifestation of healing processes up to 7 days, recovery on the 9th day § Incomplete manifestation of healing processes up to 7 days, recovery on 8 days treatment shifted and for samples 1,2 and 3 were 5 days after application of the drug. For samples 4 and 5, the therapeutic efficacy indicators are maximum, and the recovery period is 3 days (table 20). Therefore, the use of appropriate concentrations is most appropriate. 6 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

Table 15. Cows of the fifth lactation with the development of a pathological process corresponding to M3 lesions according to the dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 14 ± ** 11 79% 2 15 ± †† 12 80% 3 15 + 12 87% 4 14 + 13 93% 5 17 + 14 94% Subtotal 75 62 83% Table 16. Cows of the first lactation with the development of a pathological process corresponding to M4 lesions according to the Dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 15 + 13 87 % 2 16 + 15 94 % 3 15 + 14 93 % 4 15 + 15 100 % 5 19 + 19 100 % Subtotal 80 76 95% Table 17. Cows of the second lactation with the development of a pathological process corresponding to M4 lesions according to the Dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 16 + 15 94% 2 17 + 16 94% 3 15 + 14 93% 4 16 + 16 100% 5 14 + 14 100% Subtotal 78 75 96% Table 18. Cows of the third lactation with the development of a pathological process corresponding to M4 lesions according to the Dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 13 + 14 92% 2 14 + 13 93% 3 15 + 12 94% 4 12 + 11 92% 5 14 + 14 100% Subtotal 68 64 94% Table 19. Cows of the fourth lactation with the development of a pathological process corresponding to M4 lesions according to the Dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 14 + 12 86% 2 13 + 12 92% 3 11 + 10 91% 4 16 + 16 100% 5 14 + 14 100% Subtotal 68 64 94% Table 20. Cows of the fifth lactation with the development of a pathological process corresponding to M4 lesions according to the Dopfer classification. Sample number Group of animals (n) Treatment by day (from 1 to 7 days) Number of recovered Percentage of recovered 1 2 3 4 5 6 7 1 14 + 12 86% 2 16 + 15 94% 3 15 + 14 93% 4 13 + 13 100% 5 17 + 17 100% Subtotal 75 71 94% ** Incomplete manifestation of healing processes up to 7 days, recovery on 11 days †† Incomplete manifestation of healing processes up to 7 days, recovery on the 9th day 7 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

3 Conclusion 1. The Developed drug for the treatment of diseases of the distal extremities using niosomal forms of zinc oxide and copper sulfate is presented in concentrations 0,20%; 0,25%; 0,30%; 0,40%; 1,3% and available ointment base. All prepared samples had high stability, were easily applied to various surfaces of both the skin and its keratinized areas (hooves), and remained unchanged on the surfaces for a long time. 2. Control tests were Conducted to study the effectiveness of drug samples in concentrations 0,20%; 0,25%; 0,30%; 0,40%; 1,3% on sick cattle with various degrees of damage to the distal extremities. It was found that with the development of the intensity of the pathological process (from M1 to M4), the number of recovered animal’s decreases M4 - 94.6±2.0%; M1 – 94.2±3.8%; M2 – 86.4±11%; M3-84.0±3.0% due to the aggravation of the severity of the infectious process localized in the distal limb. The most curable were sick animals of the first and second lactation, regardless of the concentration of the samples used. References 1. A. Gomez, N.B. Cook, J. Rieman, K.A. Dunbar, K.E. Cooley, M.T. Socha, D. Döpfer, Journal of Dairy Science, 98, 927-936 (2015) 2. Floor Biemansa, Piter Bijma, Natasja M.Bootsa Mart C.M.de Jonga, Journal Epidemics, 23, 76-84 (2018) 3. R. Cheli and C. Mortellaro 8th International Conference on Diseases of Cattle, Piacenza, Milan, Italy, 208-213 (1974) 4. I.Demirkan, L.Walker, D.Murray, W.Blowey, The Veterinary Journal, 157, 69-77 (1999) 5. Deryck H. Read, Richard L. Walker, J. Vet. Diagn. Invest., 10, pp. 67-76, (1998) 6. Schrank K, Choi BK, Grund S, Moter A, Heuner K, Nattermann H, GobelUB. Int. J. Syst. Bacteriol. 49, 43–50 (1999) 7. Evans NJ, Brown JM, Demirkan I, Singh P, Getty B, Timofte D, Vink WD, Murray RD, Blowey RW, Birtles RJ, Hart CA, Carter SD. J. Clin. Microbiol. 47, 689–696 (2009) 8. Yano T, Yamagami R, Misumi K, Kubota C, Moe KK, Hayashi T, Yoshitani K, Ohtake O, Misawa N. J. Clin. Microbiol. 47, 727–733 (2009) 9. Wilson-Welder, J. H., M. K. Elliott, R. L. Zuerner, D. O. Bayles, D. P. Alt, and T. B. Stanton. BMC Microbiol, 13, 280 (2013) 10. Gomez, A., K. S. Anklam, N. B. Cook, J. Rieman, K. A. Dunbar, K. E. Cooley, M. D. Socha, and D. Döpfer. J. Dairy Sci. 97, 4864–4875 (2014) 11. Nascimento, L. V., M. T. Mauerwerk, C. L. Dos Santos, I. R. Barros Filho, E. H. Birgel Júnior, C. S. Sotomaior, H. M. Madeira, and R. D. Ollhoff. J. Clin. Microbiol. 53, 1935–1937 (2015) 12. Evans, N. J., J. M. Brown, I. Demirkan, P. Singh, B. Getty, D. Timofte, W. D. Vink, R. D. Murray, R. W. Blowey, R. J. Birtles, C. A. Hart, and S. D. Carter. J. Clin. Microbiol. 47, 689–696 (2009b) 13. Blowey, R. W., and M. W. Sharp. Vet. Rec. 122:505– 508 (1988) 14. Döpfer, D., A. A. H. M. ter Huurne, J. L. Cornelisse, A. J. A. M. van Asten, A. Koopmans, F. A. Meijer, Y. H. Schukken, I. Szakall, W. Klee, and R. B. Bosma.. Vet. Rec. 140, 620–623 (1997) 15. Klitgaard, K., Boye, M., Capion, N., Jensen, T.K., J. Clin. Microbiol. 46, 3012–3020 (2008) 16. Rasmussen, M., Capion, N., Klitgaard, K., Rogdo, T., Fjeldaas, T., Boye, M., Jensen, T.K., Vet. Microbiol. 160, 151–161 (2012) 17. Döpfer, D., Anklam, K., Mikheil, D., Ladell, P., Vet. J. 193,685–693 (2012a) 18. Al-Qudah, A., Mostratos, A., Quesnel, L. J. Appl. Bacteriol. 55, 417–428 (1983) 19. Mumba, T., Kruitwagen, C., Dreher, M., Gaastra, W., van der Zeijst, B. J. Vet. Med. Ser. B 46,117–126 (1999) 20. Laven, R., Proven, M. Vet. Rec. 147, 503–506 (2000) 21. Speijers, M., Baird, L., Finney, G., McBride, J., Kilpatrick, D., Logue, D., J. Dairy Sci. 93, 5782– 5791 (2010) 8 BIO Web of Conferences 30, 06001 (2021) https://doi.org/10.1051/bioconf/20213006001 ILS 2020

Development of approaches to the diagnosis of cattle leukemia in the system of antiepizootic measures in the Belgorod Region Anatoly Kovalenko1* , Irina Donnik2 , Andrey Dorofeev1 , Svetlana Belyaeva1 , Nazar Yavnikov1 , Victoria Oskolskaya1 , Danila Karaychentsev3 , and Maxim Petropavlovskiy4 1Belgorod State Agrarian University named after V. Ya. Gorin, vil. Mayskiy, Russia 2Russian Academy of Sciences, Moscow, Russia 3Veterinary Department of the Belgorod region, Belgorod, Russia 4Ural Federal Agrarian Research Center of the Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia Abstract. The aim of the research is the use of serological and molecular genetic methods for detecting virus-infected cattle leukemia, as well as determining the significance of PCR in identifying BLV infected calves in the system of antiepizootic health measures. The developed technique for early diagnosis of leukemia in cattle made it possible to accelerate the process of recovery of disadvantaged farms in the Belgorod region by increasing the frequency of studies from 6 months to 2-3 months and an increase in the sensitivity of the agar-gel immunodiffusion test. This, in turn, leads to an increase in the sensitivity of the agar-gel immunodiffusion test and makes it possible to detect, on average, from 8.8% to 20.25% more animals infected with the leukemia virus compared to the standard reaction of the agar-gel immunodiffusion test. The additional use of molecular genetic tests for the detection of proviral DNA of the leukemia virus makes it possible to identify at the early stages of the development of the leukemia process, in calves from 15 days of age, the genomic material of bovine leukemia virus, which will also allow in a shorter time to carry out a qualitative improvement of young cattle in dysfunctional farms. 1 Introduction Viral infectious diseases of animals continue to occupy a dominant position in the system of antiepizootic measures carried out at livestock agricultural enterprises of the Belgorod region. One of the most pressing diseases in this case is cattle leukemia (EBL) both in the Belgorod region and in most countries of the world, causing a high level of infection in livestock and the associated economic costs of antiepizootic measures. A rather late detection of infection in animals, despite the use of modern diagnostic tests, contributes to the wide spread of leukemia in cattle [1, 2]. Modern requirements for recovery from bovine leukemia are in need of early detection of virus-infected animals in the early stages of development of the incubation period. Ways to solve this problem require the development of new possibilities in the diagnosis and prevention of viral infectious diseases of animals in the system of antiepizootic measures. Bovine leukemia (EBL), caused by B-lymphotropic leukemia virus (BLV), belongs to the RNA-containing viruses of the Retroviridae family, the genus Deltaretrovirus, is a slowrunning chronic infectious disease of a tumor nature [3, 4, 5]. Leukemia is still one of the most economically significant diseases, causing significant damage to the development of dairy and beef cattle breeding, and also has a potential danger to humans [6-15]. The efficiency of detecting sick animals at earlier stages of development of the incubation period with slowly progressing infections * Corresponding author: [email protected] is the key to the formation of stable well-being in cattle leukemia. The process of effective recovery of cattle from leukemia was significantly accelerated due to the use of developed serological diagnostic test systems that facilitate the rapid identification of infected individuals during the implementation of health-improving and preventive measures [16]. The basic serological test for detecting infected cattle leukemia virus is the reaction of the agar-gel immunodiffusion test followed by the use of a hematological test [17]. In addition, in recent years, the method of molecular genetic analysis (polymerase-chain reaction) has been increasingly used to diagnose bovine leukemia. The high specificity of this method is achieved due to the detection of a fragment of the leukemia virus proviral DNA. The specificity is determined by the nucleotide sequence of the primers, which eliminates the possibility of false results. The polymerase-chain reaction method has high sensitivity, making it possible to detect single fragments of viral nucleic acids [18]. The difficult epizootic situation of bovine leukemia in the Russian Federation, including in the dairy enterprises of the Belgorod region, served as the beginning of research on the early detection of infected animals in dysfunctional farms in order to increase the success of the anti-leukemic health measures. The aim of the research is the use of serological and molecular genetic methods for detecting virus-infected © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 06002 (2021) https://doi.org/10.1051/bioconf/20213006002 ILS 2020

cattle leukemia, as well as determining the significance of the polymerase chain reaction in identifying BLV infected calves in the system of antiepizootic health measures in the Belgorod region. The subject of the research is epizootic screening for bovine leukemia in the Belgorod region and the development of approaches to early diagnosis of bovine leukemia. The object of the study is dairy cattle, animals infected and sick with leukemia. 2 Materials and methods The research was carried out on the basis of the Belgorod State Agrarian University (testing laboratory, laboratory for the study of infectious and invasive diseases and approbation of veterinary drugs); Shebekinskaya interdistrict veterinary laboratory; using the data of the Belgorod Veterinary Directorate on the epizootic state in the dairy farms of the Belgorod region with leukemia - LLC MMF Nezhegol and JSC Orlik p. Voskresenovka, Chernyansky District; CJSC “Voskhod” and LLC “Pobeda” of Shebekinsky district. Research methods - epizootological, serological (of the agar-gel immunodiffusion test), hematological and molecular biological (polymerase chain reaction) research methods. The material for the research was epizootological data on the prevalence of bovine leukemia, as well as blood serum and stabilized blood from cattle in dairy farms of prosperous and dysfunctional farms for bovine leukemia. Blood samples were taken with disposable instruments into sterile tubes from the tail vein to obtain blood serum for studies in the agar-gel immunodiffusion test, as well as stabilized blood for studies by the hematological method and polymerase chain reaction. The studies were carried out according to the approved methods: - methodology for epizootological examination of cattle (Order of the Ministry of Agriculture and Food of the Russian Federation of 11.05.1999 N 359 “On the approval of the rules for the prevention and control of leukemia in cattle”, registered in the Ministry of Justice of the Russian Federation on 04.06.1999 N 1799); - methodology for conducting hematological, serological studies and molecular biological methods (Guidelines for the diagnosis of bovine leukemia, approved by the head of the Veterinary Department of the Ministry of Agriculture of the Russian Federation M.V. Kravchuk on 08/23/2000, N 13-7-2 / 2130) ; - hematological studies of the blood of animals were carried out on an automatic hematological analyzer URIT-3020 (China) according to the instructions for working with this device. Visualization of hematological parameters on a glass slide after Leishman staining was carried out on a Labor microscope, 60X/ 0.75 with a built-in camera. Counting leukocytes in the Goryaev chamber according to the method of I.P. Kondrakhin - Molecular genetic studies (polymerasechain reaction) - used the test system “Leukemia” according to the instructions for its use, approved by the head of the Rosselkhoznadzor dated 19.05. 2009 [18]. Statistical processing of the data was carried out by the methods of variation statistics in the form of calculating the arithmetic mean, standard deviation and coefficients of variation using the Microsoft Office Excel 2007 software. 3 Results and discussion The epizootic situation in bovine leukemia has remained unchanged in the Russian Federation over the past few years. Thus, the number of cattle most infected with BLV is in the Central Federal District and is more than 41%. (Fig. 1). Federation conducted retrospective and prospective monitoring epizootic studies to study the epizootic situation of BLV infection in dairy farms in the Belgorod region showed that prior to the start of the rehabilitation plan for cattle leukemia, the situation remained tense in the region. It should be noted that in most farms belonging to large holdings, the level of infection with leukemia in cattle is significantly lower in comparison with dairy farms containing aboriginal cattle for a long period of lactation Fig.1. Study of the epizootic situation of cattle leukemia in the Russian. This is primarily due to the fact that in dairy holdings, animals are exploited a small number of lactations, on average from 2 to 4, and since this infection is slow, it is possible to detect the presence of anti-leukemia antibodies in such animals using the agar-gel immunodiffusion reaction by 2-3 lactation. At the beginning of the year, in the dairy farms of the Belgorod region, the farms of the Shebekinsky and Chernyansky districts of the region were mostly unfavorable for leukemia in cattle, in which there were a significant number of the agar-gel immunodiffusion (+) animals - up to 85%. The developed approach to the diagnosis of bovine leukemia in the system of antiepizootic measures in the Belgorod region, in dysfunctional farms, is based on a two-fold reduction in the frequency of serological studies and an increase in the desired serum antiviral antibodies in the test samples during immunodiffusion reactions. So, at the beginning of the year in the Belgorod region, there were only four farms unfavorable for leukemia in cattle - LLC MMF Nezhegol (dairy farm No. 1), JSC Orlik (dairy farm No. 2), CJSC Voskhod (dairy farm No. 3 ) and LLC “Pobeda” (dairy farm No. 4) of the Belgorod region - with an infection rate from 31.7 to 84.1%, and at the end of the year these indicators decreased, respectively, from 11.5 to 80.2% as a result of the developed approach to diagnostics of cattle leukemia in the system of antiepizootic measures in the Belgorod region (Fig. 2). 2 BIO Web of Conferences 30, 06002 (2021) https://doi.org/10.1051/bioconf/20213006002 ILS 2020

At the beginning of 2020, in the Belgorod region, dairy farm Nezhegol LLC and Orlik AO s. Voskresenovka of Chernyansky district (dairy farm No. 1 and No. 2); Voskhod CJSC and Pobeda LLC of Shebekinsky District (dairy farm No. 3 and No. 4), and at the end of the reporting period of 2020, it was possible, using the approaches of early diagnosis of bovine leukemia, improving the farm health improvement system, to fully improve the livestock of Orlik JSC (dairy farm No. 2). In general, in the Belgorod region as of January 2020, taking into account the unfavorable conditions of leukemia in cattle among the four abovementioned farms with a livestock of 6605 heads, 3343 heads were identified as react in the agar-gel immunodiffusion test, which was 50.6%. At the same time, hematologically sick individuals were identified 219 heads (6.55%) (Fig. 3). At the end of 2020, the use of the developed technique for early diagnosis of bovine leukemia in order to improve the health improvement system made it possible to increase the detection rate of infected livestock in the agar-gel immunodiffusion test from 1668 to 1741 heads, which was 6.35% more than at the beginning of the study period. As for the isolation of animals with hematological leukemia in cattle, at the end of the year their number was only 22 heads, which in percentage terms was 1.26%. This is 5.2 times less than at the beginning of the year. To test the improved methodology for setting up the immunodiffusion reaction in agar-gel, 1251 samples of cattle blood serum were taken from a dysfunctional leukemia farm - Pobeda LLC. In the experimental part of the research, various centrifugation methods and temperature regimes were used for these samples of animal blood serum in order to identify the maximum number of additional of the agar-gel immunodiffusion (+) of bovine heads that previously gave negative immune diffusion reactions in the standard setting of the agar-gel immunodiffusion test. As a result of the studies, a direct relationship was established between the concentration of the desired antibodies in the tested sera (high-speed centrifugation) and the absolute binding of antibodies to antigens under conditions of the reaction that are more physiologically close to in vivo (37ºC). This leads to an increase in the sensitivity of the agar-gel immunodiffusion test, and also makes it possible to detect, on average, 20.25% more animals infected with the leukemia virus compared with the standard agar-gel immunodiffusion test. The use of the proposed method for the diagnosis of leukemia in cattle makes it possible to identify animals infected with leukemia at earlier stages, which are in the initial period of the production of antileukemic antibodies, which makes it possible to reduce the time they spend in the herd, and this ultimately helps to accelerate the implementation of health-improving measures without requiring significant material and time costs. In the complex of diagnostic studies, as an additional research method, a polymerase chain reaction was used using the Leukemia test system on the livestock of Pobeda LLC calves obtained from the agar-gel immunodiffusion of (+) animals. In a pilot study in January 2020, 22 samples of clinical material from young animals up to 2 months and in the maternity ward - calves up to 3 weeks - 3 heads tested positive for the presence of bovine leukemia virus. Further, a study was carried out of the total number of calves from 15 days to 6 months, obtained from the IDR (+) cows (table I). As a result of the studies, in January, from 114 calves (blood samples) in 16 samples, the presence of bovine leukemia virus was detected, which amounted to 11.11%. These data provided the rationale that in subsequent studies conducted in May-June, this test was also used on calves in order to prevent the spread of leukemia through infected young animals. Since these individuals are sources of the causative agent of infection and, being in a common herd with other calves, they infect the rest of the livestock with the leukemia virus. In order to avoid and to exclude the presence of this mechanism of transmission of leukemic infection, subsequent studies using polymerase chain reaction were carried out every three to four months. Studies carried out in May of blood samples from 50 head of calves, from 15 days to 3 months, revealed the presence of bovine leukemia virus in 14 samples, i.e., in 28% of samples received from this livestock. Later, in June, already in the study of calves from 15 days to 1.5 months, only 2 samples were identified out of 36 blood samples, in which the bovine leukemia virus was present, which was 5.5% of the total number of animals studied. In general, during the study period, the presence of leukemia virus was detected in 32 calves up to 6 months of the total number of 230 calves obtained from the reaction of the agar-gel immunodiffusion (+) cows, which amounted to 13.9% (table I). All calves in which the proviral DNA of the leukemia virus was detected in the blood were removed from the general herd and fed in a separate room for removal after gaining the appropriate weight from the farm. Thus, molecular genetic studies using the polymerase chain reaction of the Leukemia test system allowed identifying 32 infected calves, which amounted to 13.91%. In the system of antiepizootic measures to accelerate the recovery of dysfunctional farms, we proposed to reduce the frequency of serological studies from 6 to 3 months. Since a six-month period of research can contribute to reinfection, the agar-gel immunodiffusion (-) of animals with infected individuals in the early stages of the development of the leukemia process, in which antibodies to bovine leukemia virus have not previously been detected in the agar-gel immunodiffusion test and which had a low antiviral antibody index. The developed approach to the diagnosis of leukemia in cattle during the implementation of healthimproving measures made it possible to fully recover from leukemic infection the number of cattle dairy farm JSC “Orlik” p. Voskresenovka, Chernyansky District (dairy farm No. 2), and in the other two previously unsuccessful farms for leukemia: CJSC Voskhod and OO Pobeda (dairy farm No. 3 and No. 4), in Shebekinsky District, Belgorod Region, reduce the infection rate from 56.4 % and 84.1% to 41.4% and 11.5%, respectively (table II). Analyzing the data in table II, it should be noted that the most successful anti-leukemic measures using the developed approach to diagnostics were at CJSC “Voskhod” (dairy farm No. 3). In this farm, at the 3 BIO Web of Conferences 30, 06002 (2021) https://doi.org/10.1051/bioconf/20213006002 ILS 2020

beginning of the year, before the use of early diagnosis, there were 56.4% of infected individuals, and at the end of the study period, 41.4%, i.e., the number of infected animals decreased by 15%, and the excretion rate of hematologically sick animals decreased from 12.3% to 1.9%, i.e., 6.5 times (table II, Fig. 6). In OOO “Pobeda” (dairy farm No. 4), the rate of leukemia infection in cattle decreased from 84.1% to 11.5%, i.e. by 72.6%, however, the number of hematologically sick animals increased intensively from 3.5% to 16.4% (table I, Fig. 7). At OOO Nezhegol (dairy farm No. 1), the process of more active detection of leukemia-infected and hematologically sick animals is observed through the use of early diagnosis. This is manifested by an increase in the number of infected and hematologically sick animals by 17% and 3.5%, respectively. In our opinion, this is due to the presence of a larger number of animals, in comparison with previous farms, which are in the latent stage of the leukemia process with a low antibody index, which in turn affects the results of the agar-gel immunodiffusion test (table I, Fig. 4). Thus, it is necessary to note the application of the developed approaches to the early diagnosis of bovine leukemia when carrying out health-improving antiepizootic measures made it possible to completely recover from leukemic infection the number of cattle in the dysfunctional dairy farm Orlik JSC (dairy farm No. 2) (table I, Fig. 5), and in the other two previously unsuccessful farms for leukemia: CJSC “Voskhod” and OO “Pobeda” to reduce the level of infection, respectively, from 56.4%, 84.1 to 41.4%, 11.5%. The effectiveness of the used early diagnosis of bovine leukemia when carrying out antiepizootic health measures in these two dysfunctional farms is evidenced by the data on 2 and 3.6 times decrease in the infection rate of susceptible livestock of animals, respectively, in dairy farm No. 3 and dairy farm No. 4 (Fig. 6-7). In general, on all dysfunctional farms, due to the impeccable implementation of the plan for the implementation of the program to improve the recovery of the Belgorod region from cattle leukemia, it was possible to reduce the level of infection of the livestock of BLV animals by half (Fig. 8). Table 1. Results of the study of blood samples from calves of LLC “POBEDA” in the polymerase chain reaction using the “leukemia” test system № Total inspected calves (n) Age of animals Number of positive samples in PCR % to the total number of tested samples in the PCR Note January 1 144 15 days – 6 months 16 11,11 Number of heifers for the study period May 2 50 15 days – 3 months 14 28,0 Number of heifers born during the study period June 3 36 15 days – 1,5 months 2 5,55 Calves from (+) IDR cows TOTAL 230 - 32 13,91 - Table 2. Dynamics of changes in the level of infection and the number of hematologically sick animals in farms with poor leukemia in cattle on the territory of the Belgorod region for 2020 № Farm name Total livestock, heads Infection rate of animals, % Number of the + IDR animals, heads Number of revealed hematologically sick animals, heads / % 1. Before using early diagnosis dairy farm №1 1105 67,7 748 59/7,9 After a year of using early diagnosis dairy farm №1 1090 80,2 874 100/11,4 2. Before using early diagnosis dairy farm №2 3526 31,7 1118 75/6,7 After a year of using early diagnosis dairy farm №2 572 0 0 0 3. Before using early diagnosis dairy farm №3 663 56,4 374 46/12,3 After a year of using early diagnosis dairy farm №3 1637 41,4 678 13/1,9 4. Before using early diagnosis dairy farm №4 1311 84,1 1103 39/3,54 After a year of using early diagnosis dairy farm №4 1009 11,5 116 19/16,4 TOTAL 10913 59,98/33,28 835,75/417,0 54,75/33,0; 7,61/7,43 4 BIO Web of Conferences 30, 06002 (2021) https://doi.org/10.1051/bioconf/20213006002 ILS 2020

Fig.5. The dynamics of changes in the level of infection and the number of hematologically sick animals in a farm with leukemia in cattle on the territory of the Belgorod region in 2020 (dairy farm No. 2) Fig.4. The dynamics of changes in the level of infection and the number of hematologically sick animals in a farm with leukemia in cattle on the territory of the Belgorod region in 2020 (dairy farm No. 1). Fig.3. The results of the use of the method of early diagnosis of leukemia in cattle in the recovery of leukemia-dysfunctional farms in the Belgorod region. Fig.2. Dynamics of the level of infection with leukemia virus in the number of cattle in stationary leukemiadysfunctional farms of the Belgorod region. Fig. 6. The dynamics of changes in the level of infection and the number of hematologically sick animals in a farm with leukemia in cattle on the territory of the Belgorod region in 2020 (dairy farm No. 3). Fig. 7. The dynamics of changes in the level of infection and the number of hematologically sick animals in a farm with leukemia in cattle on the territory of the Belgorod region in 2020 (dairy farm No. 4). Fig. 8. Dynamics of changes in the level of infection and detection of hematopoietic patients after the use of early diagnosis of leukemia in cattle during the implementation of recreational activities in dysfunctional farms in the Belgorod region. 5 BIO Web of Conferences 30, 06002 (2021) https://doi.org/10.1051/bioconf/20213006002 ILS 2020

4 Conclusion As a result of monitoring, retrospective and prospective epizootic studies, as well as serological, hematological and molecular genetic studies in conjunction with the state veterinary service of the Belgorod region, it was possible to reduce the level of leukemia infection of the livestock population by half in dairy farms (dairy farm No. 1, No. 2 , No. 3 and No. 4), thanks to strict adherence and implementation of the plan for the implementation of the program for the recovery of the Belgorod region from bovine leukemia. The developed technique for early diagnosis of leukemia in cattle made it possible to accelerate the process of recovery of disadvantaged farms in the Belgorod region by increasing the frequency of studies from 6 months to 2-3 months and an increase in the sensitivity of the agar-gel immunodiffusion reaction, which was accompanied by an intensive decrease in the detection rate of hematologically sick animals 5.2 times and an increase in the number of infected individuals by 6.35%. The use of early diagnosis of leukemia in cattle during the implementation of health-improving measures has made it possible to fully recover from leukemic infection the number of cattle in the dairy farm JSC “Orlik” s. Voskresenovka, Chernyansky District (dairy farm No. 2), and in the other two previously unsuccessful farms for leukemia: Voskhod CJSC and Pobeda OO (dairy farm No. 3 and No. 4) of Shebekinsky District, Belgorod Region, reduce the infection rate from 56.4% , and 84.1% to 41.4%, and 11.5%, respectively. When practicing the step-by-step detection of animals with leukemia using the developed approach to the diagnosis of cattle leukemia in the system of antiepizootic measures to enhance the health improvement system in a separate farm, OOO Pobeda, a new approach to staging the reaction of the agar-gel immunodiffusion was developed. It has been proved that the proposed method of improved the agar-gel immunodiffusion test in diagnostics of bovine leukemia is based on establishing a direct relationship between the concentration of the desired antibodies in the tested serum and the absolute binding of antibodies to antigens under more physiologically close to in vivo reaction conditions This, in turn, leads to an increase in the sensitivity of the agar-gel immunodiffusion test and makes it possible to detect, on average, from 8.8% to 20.25% more animals infected with the leukemia virus compared to the standard of agar-gel immunodiffusion test. The use of the proposed method for diagnosing leukemia in cattle makes it possible to identify animals infected with leukemia at earlier stages of the development of the leukemia process, which are in the initial period of the production of antileukemic antibodies, which makes it possible to reduce the time they spend in the herd, and this ultimately helps to accelerate the implementation of health-improving anti-leukemic measures without requiring significant material and time costs. At the same time, the additional use of molecular genetic tests for the detection of proviral DNA of the leukemia virus makes it possible to detect the genomic material of bovine leukemia virus at the early stages of the development of the leukemia process in calves from the age of 15 cattle in dysfunctional farms. References 1. M.I. Gulyukin, O.V. Kapustina, I.Yu. Ezdakova, S.V. Valtsiferova, T.V. Stepanova, M. Anoyatbekov, Problems of Virology, 64, 173-177 (2019). 2. A.De Brogniez, A.B. Bouzar, J.R. Jacques [et al.], J. Virology, 89, 8945-8956 (2015). 3. M.I. Gulyukin, N.G. Kozyreva, L.A. Ivanova, T.V. Stepanova, A.I. Klimenko, A.V. Kovalenko, Y.D. Drobin, V.N. Vasilenko, Problems of Virology, 60, 32-37 (2015). 4. A.F. Valikhov, Moloch.prom-st, 9, 74-77 (2018). 5. I.M. Donnik, I.A. Shkuratova, A.T. Tatarchuk [et al.], Regulatory issues in veterinary medicine, 2, 42– 46, 2015. 6. T.V. Stepanova, Russian J. Agricultural and SocioEconomic Sciences (RJOAS). 8, 49-56 (2016). 7. S.V. Timoshina, O.B. Badeeva, Veterinary medicine and feeding, 4, 6-7 (2012). 8. V.A. Mishchenko, O.N. Petrova, A.K. Karaulov, A.V. Mishchenko, The problem of leukemia in cattle (Vladimir: FSBI ARRIAH, 2018). 9. V.V. Khramtsov, N.G.Dvoeglazov, R.S. Khafizova, Innovations and food security, 2, 61-70 (2014). 10. G.M. Sviridenko, Dairy industry, 8, 13-16 (2017). 11. M.C. Frie, K.R. Sporer, J.C. Wallace, R.K. Maes, L.M. Sordillo, P.C. Bartlett [et al.], Vet. Immunol. Immunopathol, 182, 125-135 (2016). 12. S. Suzuki, S. Konnai, T. Okagawa, R. Ikebuchi, A. Nishimori, J. Koharaetal, Vet. Immunol. Immunopathol, 163, 115-124 (2015). 13. H. Murakami, H. Todaka, J. Uchiyama, R. Sato, K. Sogawa, M. Sakaguchiet [et al.], Virology, 537, 45- 52 (2019). 14. The effect of the drug “Leukozav” on the cellular component of immunity in rats: EESJ electronic journal: [site]. URL: https://eesajournal.com/2017/03/14/vliyanie-preparatalejkozav-na-kletochnoe-zvo 15. I.Yu. Ezdakova, O.V. Kapustina, M.I. Gulyukin, T.V. Stepanova, Problems of Virology, 65, pp. 35- 40 (2020). 16. I.M. Donnik [et al.], (Yekaterinburg: Scientific advice, THEM. IRA UTK LLC, 2011). 17. I.M. Donnik, G.A. Jailidi, S.V. Tikhonov, Veterinary of the Kuban, 5, 15-19 (2013). 18. Instructions for the use of the test system “leukemia” for the detection of leukemia virus in cattle (cattle) by the polymerase chain reaction, approved May 19, 2009. [Electronic access]. 6 BIO Web of Conferences 30, 06002 (2021) https://doi.org/10.1051/bioconf/20213006002 ILS 2020

Participation of dopaminergic system in the dynamics of behavioral reactions of aging rats of both sexes under daily testing in the open field Elena Birukova 1* , Denis Khusainov1 , Andrew Chajka1 , Natalya Tribrat 1 , Zera Dzhemalyadinova1 , Natalia Dmitrenko1 and Svitlana Chornobay1 1 Taurida Academy, V.I. Vernadsky Crimean Federal University, Simferopol, Russia Abstract. Under daily testing of aging rats of both sexes in the open field a slow lowering of motion activity is observed: by the 6th day of the experiment the passed distance of male rats reduces by 41% (р≤0.01), and of female rats – by 46% (р≤0.05) in comparison to the background values. The blockade of monoamine oxidase-B (МАО-В) by selegiline (intraperitoneal introduction of 5 mg/kg one hour before testing) does not significantly change the orientation and intensity of this process, but probably contribute to the formation of a more comfortable psycho-emotional state of the animals. Consequently, the central dopamine cannot prevent from the motion activity lowering process of aging rats under daily testing, at least, in the frames of the experiment method used in this research. We obtained the evidence in favor of the fact that the suppression of the motion activity under daily testing is rather connected with the animals’ loss of motivation for research than with the growing anxiety. 1 Introduction It is well-known that in case of a repeated phenomenon of any modality and nature the interest to it is gradually lowering up to a complete suppression of its significance. Under the experimental laboratory conditions this suppression is manifested, for example, through lowering of such indicators as the passed distance, the exploratory activity, the time and amount of going out to the centre [1, 2]. On the whole, an animal stops exploring the testing space and demonstrates a passive behaviour. The factors, which influence the dynamics of this process, are still being actively discussed in the scientific community [2]. Thus, on the issue of the central dopamine participation there are data on the suppression of motion activity [3], as well as its stimulation [4], or even on the absence of the obvious effect [5]. The presence of antidepressant and anxiolytic effects is observed [3, 6], as well as the grooming stimulation [7, 8], or, on the contrary, the absence of meaningful psycho-emotional behavioral reactions [9]. 1.1 Purpose of the study Thus, we have put forward the hypothesis that the increase of the central dopamine concentration by blocking MAOB may quantitatively and qualitatively influence the dynamics of motion activity and the anxiety level of both sexes rats in the open field, which is caused by the repeated 6-day testing. * Corresponding author: [email protected] 2 Experimental For the experimental work 30 male rats of Wistar type (320 – 350 gr., 15.5 – 16 months) with an average level of motion activity and anxiety were selected. Further, the selected animals were divided into 3 groups: “Control” (n=10), “Placebo” (n=10) and “Selegiline” (n=10); and were left for ten days under standard conditions of the vivarium for a social adaptation. On the expiry of this period the animals from all the groups were tested daily in the circular open field with the diameter 1 m during 6 days in succession. From the vivarium the animals were taken to the ethology laboratory at 11.00, for one hour they were left alone to adapt to the laboratory conditions, then at 12.00 they were injected, and at 13.00 the test started. The rats from Group “Control” did not undergo any additional influences, the rats from Group “Placebo” were daily injected intraperitoneally by 0.2 ml of physiological solution 60 minutes before the test, the animals from Group “Selegiline” were injected the corresponding substance in the dose of 5 mg/kg in the volume of 0.2 ml. Testing of each individual lasted 5 min, and before the experiment a so called zero rat was driven around in the behavioral test. The field was thoroughly wiped with a low-alcohol solution after each animal. In the case with the female rats (270 – 300 gr., 15.5 – 16 months) all the groups and operations with them were identical to the ones with the male rats. The animal grooming in both gender groups was also analyzed. We divided this indicator into three, more or © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 06003 (2021) https://doi.org/10.1051/bioconf/20213006003 ILS 2020

less, definite forms: an interrupted grooming of separate body parts (discomfort marker), incomplete grooming and complete grooming (comfort marker). These forms are relative enough as till now there is no unanimity in the scientific community on the issue of classification and importance of grooming. In a separate experimental series, the anxiety index (AI) of animals of both sexes was studied in the testing equipment “Cruciform raised labyrinth”. The experimental mechanics of this research part was, as a matter of fact, identical to the one already described for the test “Open field”: 30 male Wistar rats (320 – 350 gr., 15.5 – 16 months) and 30 female rats (270 – 300 gr., 15.5 – 16 months) with an average level of motion activity and anxiety were selected. The selected animals were divided into 3 groups: “Control” (n=10), “Placebo” (n=10), “Selegiline” (n=10) and left under the standard vivarium conditions for a social adaptation. The further operations with the mentioned animal groups were identical to the ones, described for the groups, studied in the “Open field”. We should mention that the duration of the animals in the equipment “Cruciform raised labyrinth” was 5 minutes, and before the start of the experiment in the behavioral test, there was also a zero rat. The equipment was wiped with a low-alcohol solution after each animal. AI was calculated by the division of the time indicator of the animals being in the closed arms by the time of animals being in the open arms of the equipment “Cruciform raised labyrinth”: TCA/TOA. Therefore, the closer the estimate indicator is to zero, the less the anxiety level of animals is. All the researches are carried out in the testing complexes “Open field” and “Cruciform raised labyrinth” (Ltd “RPC Open Science”). The experimental video recordings were processed in the software program complex Noldus EthoVision. The statistical data processing was done in the software program GraphPad 7.0 using Dunn index. The results are presented as a median and 5/95% percentiles. 3 Results and discussion As it was expected, during six-day testing in the “Open field” we observed a slow suppression of animals’ motion activity of Wistar rats, which is expressed by the reduction of the passed distance by males by 41 % (р≤0.01) (Fig. 1А) and the falling of movement speed by 36% (р≤0.01). The passed distance by female rats was reduced by 46% (р≤0.05) (Fig. 1В), and the movement speed – by 39% (р≤0.01). All the indicators comparisons here and further are presented in relation to the 1st day. The analysis of time of two control animal groups in different zones of the “Open field” revealed that despite the sex assignment the time of being in the central zones reduces, and in the wall periphery zone increases, a so called thigmotaxis is observed. For example, the time of being in the centre of the open field reduces by 84% р≤0.01) for males, and for females – by 81% (р≤0.01) (Fig. 2). In Groups “Selegiline” the dynamics of the animals’ behavioral activity differed little from the control groups, but a slight (not reaching a significant level) increase of rats’ time being in the centre of the “Open field” was observed, as well as dominance of the complete grooming even on the sixth day of testing (Table I). This fact shows a certain improvement of the psycho-emotional background of animals. In Groups “Control” and “Placebo” of both male and female rats grooming, on the contrary, gains a more evinced interrupted character. This may indicate a growing anxiety of animals. It is necessary to explain that if a definite type of grooming was scarcely distinguished and it was impossible to interpret it, then such behavioral manifestations were not fixed. That is why on some days definite types of grooming got a reference designation “absence”, i.e. their duration tended to zero. It is also necessary to mention that the dynamics of the grooming indicator did not have impressive gender differences; that is why we presented only the table with the data on male rats. In Groups “Placebo” of both sexes there are no significant differences in the analyzed behavioral indicators from the corresponding dynamics of the control groups’ animals. Even at the level of quantitative indicators these dynamics demonstrated a substantial similarity. The mentioned fact points out the absence of any significant influence of additional manipulations, such as intraperitoneal injection of a physiological solution by the syringe for insulin injections, on the rats from these groups. At the next stage we researched the AI dynamics under daily six-day testing of rats of both sexes on the equipment “Cruciform raised labyrinth”. The main working hypothesis was: in case the suppression of the motion activity under daily testing is a consequence of the animals’ anxiety level growth, then AI has to increase gradually from the 1st to the 6th day. But the AI dynamics, which was demonstrated by the animals from Groups “Control” and “Placebo” of both sexes, does not allow making an unambiguous conclusion. Thus, the male rats from Group “Control” demonstrated a reliable growth of AI by 23 % (р≤0.05) on the 3d day of the experiment and by 27 % (р≤0.05) on the 4th day in comparison to the indicator of the 1st day. On the 5th and 6th days intragroup dispersion of the AI indicator was high and did not allow revealing probable differences. Such a high dispersion occurred because these days some of the group representatives spent enough time in the open arms of the cruciform raised labyrinth, others – in the closed arms. And this preference of each animal became pronouncedly asymmetric. An analogous situation was observed in Group “Placebo” of the male rats with the difference that AI was reliably higher than the values of the 1st day on the 4th and the 6th days (by 24% and 32% at р≤0.05 correspondingly). 2 BIO Web of Conferences 30, 06003 (2021) https://doi.org/10.1051/bioconf/20213006003 ILS 2020

Fig. 1. The change of the passed distance in the “Open field” under daily testing of male rats (А) and female rats (В) in control groups. Notes: the values of the median and 5%/95% percentiles are presented, * – the differences from the indicators of the 1st day at р≤0.05, ** – the differences from the indicators of the 1st day at р≤0.01, *** – the differences from the indicators of the 1st day at р≤0.001. Fig. 2. The thermal maps of male rats and female rats movement in the open field in the control groups. Note: the intensity of colouring and transition to warm colours is in direct proportion to the time of animals being in the field space. In the group of male rats “Selegiline” AI remained stable enough during the first four days of the research, but by the 5th day of testing the animals of this group started actively jumping off the open arms of the cruciform raised labyrinth down the floor; and this did not allow doing an adequate analysis of the data. The existence of this fact is an extremely important manifestation which indicates a low anxiety level of the animals of this group. The male rats of Group “Selegiline” start demonstrating practically fearlessness of height which is the main limit in the cruciform raised labyrinth and provides the methodological efficiency of this equipment. Therefore, in connection with the described situation, we considered unreasonable to do a comparison between AI dynamics of different groups of male rats. The second most important meaningful point of the described experimental fact is that the suppression of the motion activity of rats under daily testing in the open field is unlikely to be explained by the growing anxiety of animals. The behavioral indicators of the female rats in the cruciform raised labyrinth were noticeably more stable – in Group “Selegiline” AI was stable enough, and only one case of jumping down the floor was registered on the 5th day of the research. The AI dynamics of Groups “Control” and “Placebo” was identical: the indictor grew on the 3d day and the 6th day of the research; and there were no reliable differences on any of these days. It is not difficult to foresee that the reliable differences in the AI dynamics of the female rats will be registered on the 5th and 6th days of the research between Group “Selegiline” and Group “Control”, including “Placebo”. The comparison of AI dynamics between the female rats from Groups “Control” and “Selegeline” is presented in Fig. 3. Fig. 3. Comparison of the anxiety index of the female rats from Groups “Control” ( ) and “Selegiline” ( ) under daily testing. Notes: # – the differences between the IA values of Groups “Control” and “Selegiline” at р≤0.05; the rest signs are the same as in Fig. 1. Thus, according to the results of the six-day testing of the female rats there was no obtained unambiguous answer concerning the role of anxiety in the developing suppression of the motion activity of the rats under daily testing. But, as well as for the males, it was shown that the increase of the central dopamine concentration contributes to the AI preservation at a stable level and may cause the suppression of natural fears of animals, for example, fear of height. 3 BIO Web of Conferences 30, 06003 (2021) https://doi.org/10.1051/bioconf/20213006003 ILS 2020

Table 1. Comparison of the average time of male rats’ grooming in two groups of “control” and “Selegiline” under 6-day testing in the “open field” Interrupted grooming of separate body parts. The days of testing and an average time of grooming (sec) by Groups are shown: a) Control; b) “Selegiline” and the significant difference level. 1 st day a) absent b) absent 2 nd day a) absent b) absent 3d day a) 18±1.2 b) absent (р≤0.0001) 4 th day a) 20±2.3 b) absent (р≤0.0001) 5 th day a) 22±1.8 b) absent (р≤0.0001) 6 th day a) 23±1.2 b) absent (р≤0.0001) Incomplete grooming 1 st day a) 24±1.5 b) 24±1.3 2 nd day a) 25±2.1 b) 24±1.8 3 d day a) 10±0.8 b) 15±2.6 р≤0.01 4 th day a) 5±0.4 b)10±1.2 р≤0.01 5 th day a) absent b) absent 6 th day a) absent b) absent Complete grooming 1 st day a) absent b) absent 2 nd day a) absent b) 30±3.2 р≤0.0001 3 d day a) absent b) 40±2.5 р≤0.0001 4 th day a) absent b) 44±1.8 р≤0.0001 5 th day a) absent b) 42±2.3 р≤0.0001 6 th day a) absent b) 43±3.6 р≤0.0001 4 Conclusion Therefore, the increase of the central dopamine concentration under the blockade MAO-B does not counteract the process of the motion activity lowering of the aging Wistar rats of both sexes, which was observed under daily testing, at least, in the concentration, forming after one hour of injecting the Selegiline, the blockader of MAO-B, in the dose of 5 mg/kg. This assists to form a more stable psycho-emotional background and, probably, is able, more or less, to suppress natural (instinctive) fears of animals. According to this research such suppression had gender differences and was more evinced in the male rats. Besides, it is possible to ascertain that additional influences of low intensity, in our case it is the intraperitoneal injections of the physiological solution by the syringe for insulin injections into the animals from Group “Placebo”, do not change the dynamics of the behavioral reactions of aging rats of both sexes in any way. We are inclined to interpret the results of this research as the argument for the opinion that the disappearance of the cognitive interest, and not the anxiety increase, plays the key role in the suppression of the motion activity under daily testing of animals in the open field. The following facts are in favor of this opinion: 1. AI in Groups “Selegiline” of both sexes remained stable on the background of the motion activity suppression becoming apparent. 2.The grooming indicators changes of both sexes animals in Groups “Selegiline” can be interpreted in the direction of a comfortable psycho-emotional state, but not vice-versa. 3.The AI dynamics in Groups “Control” and “Placebo” of both sexes did not demonstrate a steady growth day by day, but rather had a wavy character. 4 BIO Web of Conferences 30, 06003 (2021) https://doi.org/10.1051/bioconf/20213006003 ILS 2020

Acknowledgment The research is done on the equipment of the Center for Collective Use “Experimental Physiology and Biophysics” of V. I. Vernadsky Crimean Federal University. References 1. A. F. Yakimovskii, I.P. Pavlov Journal of Higher Nervous Activity, 61, 212-218 (2011). 2. A. V. Kalueff, Stress, anxiety and behavior. (Kyiv: Enigma, 1998). 3. E. Nowakowska, K. Kus, A. Chodera and J. Rybakowski, J Physiol Pharmacol., 52, 863-873 (2001). 4. D. F. Smith, Psychopharmacology, 50, 81-84 (1976). 5. D. Schulz, F. A. Henn, D. Petri and J. P. Huston, Neuroscience, 329, 83-92 (2016). 6. M. N. Gordon, C. D. Muller, K. A. Sherman, et al., Pharmacol. Biochem. Behav, 63, 501-506 (1999). 7. A. Barbelivien, L. Nyman, A. Haapalinna and J. Sirviö, Basic Clin. Physiol. Pharmacol., 88, 304-312 (2001). 8. S. Amiri, H. Amini-Khoei, A. Mohammadi-Asl et al., Physiology & behavior, 163, 107-114 (2016). 9. R. Pascual and S. P. Zamora-León, Dev. Neurosci., 29, 261-267 (2006). 5 BIO Web of Conferences 30, 06003 (2021) https://doi.org/10.1051/bioconf/20213006003 ILS 2020

Comparative description of the anti-inflammatory action of the food concentrate “Enoant” and resveratrol in the model of an acute exudative inflammation Natalia Tribrat1 , Elena Birukova1* , Denis Khusainov1 , Elviza Dzheldubaeva1 , Alexandr Tribrat1 , and Svitlana Chornobay1 1Taurida Academy, V.I. Vernadsky Crimean Federal University, Simferopol, Russia Abstract. The paper considers comparing the anti-inflammatory action of the food concentrate “Enoant” and a classical antioxidant – resveratrol in the doses of 20 mg/kg in the model of an acute exudative inflammatory of animals. It was proved that resveratrol had the most anti-inflammatory action; this was manifested by a considerable decrease of the increase rate of the animal paw’s size in the formalin test after 7 and 14 days of taking the antioxidant. The food concentrate “Enoant” demonstrated anti-inflammatory properties after 7 days of taking the antioxidant; this was manifested by the increase of the animal paw’s size in the formalin test, however 14 days later a moderate anti-inflammatory reaction was registered, this was manifested by the decrease of the increase rate of the animal paw’s size in the formalin test.. 1 Introduction At present an anti-inflammatory action of many antioxidants is known [1-2]. Antioxidants are able to modify the process of inflammatory reactions and to block many signal molecules, including some genetic mechanisms [3-5]. The effects of different antioxidants have different degree of anti-inflammatory intensity and tropism to different stages of chronic inflammatory process. In connection with this there is a growth of interest in using such multicomponent compositions, in which an antioxidant, and probably anti-inflammatory, action had a cumulative character. One of such polyvalent compositions is a Crimean product – the food concentrate “Enoant”, manufactured by the plant “Magarach”. The sample antioxidant with a proved anti-inflammatory action in different models of inflammation is resveratrol. The anti-inflammatory action of most antioxidants is demonstrated in the models of chronic lingering inflammation, mainly visceral, which is demanded by the needs of therapeutic practice. However, there is no data in the scientific literature on the anti-inflammatory action of antioxidants in the models of an acute inflammatory. 1.1 Purpose of the study The purpose of the study was to carry out a comparative analysis of the anti-inflammatory action of the multicomponent food concentrate “Enoant” and resveratrol in the model of an acute exudative inflammatory. * Corresponding author: [email protected] 2 Experimental The research was carried out at the Centre for Collective Use of Scientific Equipment “Experimental Physiology and Biophysics” of V. I. Vernadsky Crimean Federal University. The research was done on 40 pedigreeless white female rats at the age of 7 months weighed 250 g. Healthy animals were selected for the experiment. The animals were divided into 2 equal groups of 20 individuals in each. The animals from Group 1 took resveratrol in the isoform of trans-stereoisomer (the producer Solgar) in the dose of 20 mg/kg during two weeks. As resveratrol is poorly dissolved in water, its powder was dissolved in 1 ml of 96 % alcohol and, brought to a homogeneous state, and was added into the drinking tanks of the animals. The animals from Group 2 took daily the food concentrate “Enoant” in the dose of 20 mg/kg during two weeks. 1 ml of 96 % alcohol was added into the prepared solution. The prepared solution was added into the drinking tanks of the animals. The food concentrate “Enoant” is produced according to technological conditions, standardizing the content of phenolic substances in the product at the level of 18-20 g/dm3, controlled photocalorimetrically by the Folin-Ciocalteu reagent [TC U 00334830.018-99 the food concentrate of grapes polyphenoles “Enoant”, IW&W “Magarach”]. According to the antioxidant action the concentrate “Enoant” excels the antioxidant properties of ascorbic acid by 15 times, and antioxidant properties of blood plasma more than by 3 times [6-8]. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 06004 (2021) https://doi.org/10.1051/bioconf/20213006004 ILS 2020

To determine the anti-inflammatory action of the antioxidants all the animals were administered the formalin test before taking the antioxidants, as well as the animals were tested on the 7th day and the 14th day of taking the antioxidants. Modelling of an acute exudative inflammation was done according to the Handbook on the experimental (preclinic) study of pharmacological substances [9]. An acute inflammatory reaction was reproduced by a subplantar introduction of 0.1 ml 2% formalin solution as a water solution into the rat’s left hind paw (“the formalin test”), which was prepared before introducing by adding of 17.5 ml of distilled water into 1 ml of 37% formaldehyde solution. The size of the limb’s edema was measured by the method of water plethysmometry 50 minutes – an hour later after introducing the formalin, as exactly after the stated time an edema and a pain reaction, caused by the inflammation, develop. This is confirmed by the fact that nonsteroid anti-inflammatory drugs (in particular, ibuprofen, acetylsalicylic acid) suppress the development of this reaction [9]. The degree of the edema after the formalin introduction, proportional to the inflammatory reaction intensity, was evaluated with the help of a digital water plethysmometer LE 7500 meant for measuring small changes of the size. That is why the mentioned equipment can be used for the observation of the development of an experimentally caused inflammation reaction of the rodents and for the evaluation of the anti-inflammatory action of pharmacological drugs. The animal’s paw was put into the chamber of the plethysmometer up to the tibiotarsal joint with the further registration of the paw’s size. After that 0.1 ml of 2% formalin solution was introduced into the left hinder paw of the rat, and an hour later after the formalin injection, an iterative measuring of the paw size in the water plethysmograph was done. The involvement of the researched antioxidant into the inflammatory process regulation was judged by the degree of the increase size of the paw, expressed in the percentage to the initial size. In particular, the increase rate of the paw’s size (IR, %) was calculated by the following formula (1): IR=− *100% (1) where IR is the rate of increase of the paw’s size an hour later of the formalin injection, expressed by the percentage, PSF is the paw size, registered an hour later after the formalin injection, IPS is the initial paw size, registered before the formalin injection. In this way the increase rate, when taking the food concentrate “Enoant” by the animals – IR(Enoant), as well as when taking resveratrol – IR(res), was evaluated. The research is carried out according to the State Standard Р-53434-2009 «The principles of a proper laboratory practice» and according to the European convention for the protection of the vertebrate animals used for experimental and other scientific purposes. All the animals were kept under the vivarium conditions at the temperature 18-22оС with a natural 12- hour light-darkness cycle, a free access to water and fullfledged granulated food (State Standard 33215-2014 “The Handbook on keeping and grooming the laboratory animals. The rules for equipping the premises and procedure arrangement”). All the researches were mainly done during the day time 11.00-13.00. The evaluation of the reliability of inter-group differences of the obtained data was done by MannWhitney test. The evaluation of the reliability intergroup differences was done by Friedman test. The calculations and graphic registration of the obtained during research data were done using the programme Microsoft Excell and the software package “STATISTICA – 8.0”. 3 Results and discussion As the results of this research show, during taking antioxidants by animals the changes in the reactions of the model of an acute exudative inflammation were registered, however these differences had a differently directed character during the first week of the observation, and during the second week the observation was of a unidirectional character, but the differences differed in the degree of intensity. А. Dynamics of the anti-inflammatory action in the formalin test during taking resveratrol by animals. Before taking the researched antioxidant by the animals IR at the injection of formalin was 29%. During a daily taking of resveratrol in the dose of 20 mg/kg, a considerable decrease of the inflammatory reaction in the formalin test was observed; this was manifested through the decrease of IR edema of the distal paw section in the dynamics of two-week observation (Table 1). So, a week later after taking resveratrol the indicator IR of the paw’s size after the formalin injection was 10.8%; this is by 62.75% (р≤0,05) lower of this indicator values, registered before taking the antioxidant. Fourteen days later after taking resveratrol the level of IR continued decreasing and was 8.49%, this is by 70.72% (р≤0,05) lower in comparison with the initial data of this indicator, registered before taking the antioxidant. Thus, the pronounced decrease of the IR indicator of the animal paw’s size after injecting formalin in the dynamics of two-week’ taking resveratrol evidences of a pronounced anti-inflammatory reaction of the researched substance. According to the scientific literature data, one of the mechanisms of the nocigenic action of the formalin is the activation of TRPA1 canals, reacting to the cold in the norm and stimulating the inflammation development, correspondingly, the decrease of the response of the inflammation reaction while doing the formalin test in the dynamics of taking resveratrol evidences of the indirect through blocking TRPA1-canals anti-inflammatory mechanism of the action of the researched antioxidant. Table 1. The increase rate (IR) of the animal paw’s size in the formalin test, registered before, and also on the 7th and 14th day of taking resveratrol in the dose of 20 mg/kg Indicators Background 7 th day 14th day IR, % 29 10.8 р≤0.05 8.49 р≤0.05 Note: reliability of differences р≤0.05 by Friedman’s test. 2 BIO Web of Conferences 30, 06004 (2021) https://doi.org/10.1051/bioconf/20213006004 ILS 2020

В. Dynamics of the anti-inflammatory action in the formalin test during taking the food concentrate “Enoant” by animals. So, before taking the researched antioxidant by animals, IR of the paw after the formalin injection was 29%. As the research results showed, two-week’s taking of the food concentrate “Enoant” was followed by differently directed reactions in the model of an acute exudative inflammation, depending on the duration of taking. Thus, 7 days after taking the antioxidant, the growth of the indicator IR up to the level of 40% was observed, this is 37% р<0.05 higher of the values of this indicator, registered before taking the food concentrate (see Table 2). The growth of the indicator of IR of the animal paw’s size in the formalin test demonstrates the increase of the reaction of an acute exudative inflammation in the formalin test. But two-week’s taking of the food concentrate “Enoant” in the dose of 20 mg/kg was followed by the change of the operational sign – the lowering of the indicator IR of the animal paw’s size in the formalin test up to the level of 22 %, which is by 24.14 % р<0.05 lower than the values of this indicator, registered before taking the food concentrate; this evidences of the antiinflammatory action of the researched antioxidant during a more prolonged taking in the model of an acute exudative inflammation. Based on the obtained results, the antioxidant complex shows differently directed action in the model of an acute exudative inflammation. In the first place, a proinflammatory orientation is observed, and in the second place – an anti-inflammatory one. Probably, the proinflammatory action, registered in the initial period of the food concentrate, is conditioned by the activation of the components of the acute phase of the inflammation, aimed at the liquidation of phlogogen, including the activation of chemotaxis factors, phagocytes migration to the nidus and further lysis of phlogogene with the subsequent regeneration. Evidently, such a reaction in the model of an acute exudative inflammation is physiological and provides an effective elimination of the factor, having provoked the inflammation. The effectiveness of the proinflammatory phase is evidenced by the occurrence of the second anti-inflammatory phase, being registered during a prolonged taking of the antioxidant complex. We may assume that a successful solution to the acute inflammation prevents the inflammatory process from becoming chronic. Table II. The increase rate (IR) of the animal paw’s size in the formalin test, registered before, and also on the 7th and 14th day of taking the food concentrate “Enoant” in the dose of 20 mg/kg Indicators Background 7 th day 14th day IR, % 29 40 р<0.05 22 р<0.05 Note: reliability of differences р<0.05 by Friedman’s test. C. A comparative evaluation of the antiinflammatory action of resveratrol and the food concentrate “Enoant” in the model of an acute exudative stress. The results of the given research evidence of different mechanisms of the anti-inflammatory action of the antioxidants under research. Thus, after taking resveratrol by animals, the pronounced decrease of the inflammatory reaction was noted; this reaction was registered a week later after taking the antioxidant, and it was less expressed two weeks later after taking this substance. At the time when in the animals, taking the food concentrate “Enoant” in the model of an acute exudative stress, the growth of pro-inflammatory reaction was registered a week later, as well as a moderate lowering of the anti-inflammatory reaction two weeks later after taking the concentrate. According to the scientific literature data, the mechanism of the anti-inflammatory effect of resveratrol is implemented through the activation of different protein molecules of sirtuins. Stimulating sirtuins Sirt3 and Sirt1, resveratrol has an indirect influence on the proteins of FOXO (forkhead box O) family. These proteins are transcriptional factors, controlling the expression of genes, responsible for proliferation, differentiation, apoptosis and reaction to the external stresses. Regulating the expression of target genes, FOXO protect the organism from unfavourable external influences, activate protective mechanisms and, ipso facto, slow down the aging processes [10]. So, taking resveratrol is accompanied by the anti-inflammatory action due to the lowering of the expression of the nuclear transcriptional factor cВ (NF-cВ), participating in the transcription of the anti-inflammatory genes, encrypting cytokines and adhesion molecules [5,11]. The chemical composition of the food concentrate “Enoant” is versatile. Thus, examining the composition of the polyphenol product, we may mention the presence of a number of monomeric flavonoids, such as cvercetin, rutin and their derivatives, such as catechine, epicatechin, epichatechin gallate. Both rutin and cvercetin have not only a pronounced antioxidant action, but also an antiinflammatory action, this allows using them in the therapy of an amount of chronic inflammatory diseases. The antiinflammatory rutin properties are manifested in the suppression of the transcription of pro-inflammatory cytokines [12]. Besides, rutin inhibits metabolism of arachidic acid – the predecessors of inflammation mediators [13], levels the synthesis of tumor’s necrosis factor [14]. The anti-inflammatory properties of cvercitine are demonstrated through the suppression of inflammation mediators synthesis, anti-inflammatory cytokines [15], through the blockade of synthesis leukotrienes, inhibition of phospholipase А2 [16], activity lowering of inducible NO-synthase, inhibition of the acute inflammation phase, as a consequence of apoptosis suppression [17]. Besides, the composition of the food concentrate “Enoant” includes monomeric flavonoids (their concentration exceeded the same, registered in the wine of “Cabernet” type, approximately by 6 times). The main part of monomeric anthocyanins is presented by malvidin glycoside and its derivatives. Thereby, the food concentrate “Enoant” is a complex food concentrate, in which many of its components can work in synergism. 3 BIO Web of Conferences 30, 06004 (2021) https://doi.org/10.1051/bioconf/20213006004 ILS 2020

Fig.1 The dynamics of the anti-inflammatory action rate expressed in % in comparison with the initial data of this indicator in the model of the acute exudative inflammation, registered in the animals while taking resveratrol and the food concentrate “Enoant” in the dynamics of two-week’s observation. Note: reliability of differences* - р<0.05 by Mann-Whitney test Thus, it is possible to conclude that resveratrol inhibits sharply the activity of anti-inflammatroy reactions and signal inflammatory molecules, while the food concentrate “Enoant” meditates its anti-inflammatory action through the activation of pro-inflammation, this is reasonable for phlogogene liquidation, after that an antiinflammatory action is demonstrated. We may suggest that the anti-inflammatory mechanism of the food concentrate “Enoant” action is more physiological and soft. 4 Conclusion 1. The anti-inflammatory effects of resveratrol and the food concentrate “Enoant” in the equivalent doses of 20 mg/kg in the model of acute exudative inflammation are shown. 2. Taking resveratrol in the dose of 20 mg/kg contributed to the reduction of the inflammatory reaction, this was manifested through the decrease of the increase rate of paw’s size in the formalin test model by 62.75% (р≤0.05) after 7 days of taking the antioxidant and by 70.72% (р≤0.05) after 14 days of taking the antioxidant in comparison with the values of this indicator, registered before the taking. Taking the food concentrate “Enoant” in the dose of 20 mg/kg contributed to the intensification of the inflammatory reaction, this was manifested through the growth of the increase rate of paw’s size in the formalin test model by 37 % (р≤0.05); after 14 days of taking the antioxidant the reduction of the inflammatory reaction was noticed, this was manifested through the decrease of the increase rate of paw’s size in the formalin test model by 24.14% (р≤0.05) in comparison with the values of this indicators, registered before taking the antioxidant. References 1. O.V. Azarova, L.P. Galaktionova, “Flavonoids: mechanism of the anti-inflammatory action”, Chemistry of vegetable raw material, 4, 61–78 (2012). 2. Yu.S. Tarakhovsky, Yu.A. Kim, B.S.Abdrasilov, E.N. Muzafarov. (Pushchino: Synchrobook, 2013). 3. N.K. Zenkov, V. E. Lankin, E.B. Menshikova, Oxidizing stress (Moscow, Science, 2001). 4. E.B. Menshikova, V.O. Tkachev, N.K. Zenkov, Molecular biology, 44, 389–404 (2010). 5. H. Ghanim, C.L. Sia, K. Korzeniewski et al., J Clin Endocrinol Metab., 96, 1409–1414 (2011). 6. Yu.A. Ogai, Magarach. Wine-growing and winemaking, 1, 37-38 (2000). 7. Yu.A. Ogai, E.A. Slastya, “Wine-growing and winemaking, 1, 25-26 (2003). 8. V.M. Semyonov, A.M. Yarosh, Ukrainian biochemical journal, 57, 50-52 (1985). 9. R.U. Khabriev, Handbook on the experimental (preclinic) study of new pharmacological substances (Medicine, Moscow, 2005). 10. S. Das, G. Mitrovsky, H.R. Vasanthi et al. Oxid Med Cell Longev, 2, 345105 (2014). 11. R.H. Wong, P.R. Howe, J.D. Buckley et al., Nutr Metab Cardiovasc Dis., 3, 23-36 (2015). 12. M. Inam, M. Altinisik, M. D. Bilgin, Cel. Biochem. Funct., 20, 291-296 (2003). 13. H. P. Kim, I. Mani, V. A. Ziboh, Prostaglandins Leukot. Essent. Fatty Acids, 58, 17-24 (1998). 14. C. C. Chuang, K. Martinez, G. Xie, et al., Am. J. Clin. Nutr., 92, 1511 – 1521 (2010). 15. G.P. Nykytyuk, M.M. Bidyuk, Clinical and experimental pathology, 2, 47-50 (2003). 16. S. Manuel, G. Milagros, V. Rocio et al., Journal of hypertension, 24, 75-84 (2006). 17. M. Chiesi, R. Schwaller, Biochem. Pharmacol., 49, 495-501 (1992). 4 BIO Web of Conferences 30, 06004 (2021) https://doi.org/10.1051/bioconf/20213006004 ILS 2020

Instrumental diagnostic methods applied for orchiectomy in cockerels Svetlana Shambazova 1* 1 Agricultural Univerisity named after V.Gorin. vil. Mayskiy, Russia Abstract. Any common surgical procedure, including orchiectomy in cockerels, requires perfect knowledge in topography and syntopy of the organ the surgery is performed on. This also implies acquiring the necessary information via the application of a number of instrumental methods such as diagnostic roentgenography and diagnostic sonography. Both methods have positive (intravital, painless and quick procedure) as well as negative (possible flaws in diagnostics due to age peculiarities) aspects. 1 Introduction Any surgical intrusion into the organism of an animal has to be flawlessly performed, which requires subspecialization and knowledge of species and age peculiarities in the topography and syntopy of the treated organs and, hence, can ultimately guarantee a successful procedure. As of yet, the data available on the age peculiarities of skeletopy of the domestic fowl classified as Dominant CZ hybrids is incomplete; therefore, the research is focused on the intravital diagnostics of the genital glands of cockerels as such that is mandatory before the surgical procedure as well as on the comparison of the obtained results and the data from the alternative researches. 2 Experimental The research was carried out in the Center of Innovative Veterinary Medicine on the premises of the Federal State Budgetary Educational Institution of Higher Education “Belgorod State Agricultural University named after V. Gorin”. The study was performed on the 120-, 90- and 60- day-old Barred D 959 cockerels. The two complementary intravital diagnostic methods included diagnostic roentgenography and diagnostic sonography. The images were described according to the NAV (The Nomina Anatomica Veterinaria) standards [1]. The Federal State Budgetary Educational Institution of Higher Education “Belgorod State Agricultural University named after V. Gorin” is licensed to work with the sources of ionizing radiation according to Part 2, Article 12 of the federal law “On Licensing Certain Types of Activities”. The roentgenography room in the Center of Innovative Veterinary Medicine of the Federal State Budgetary Educational Institution of Higher Education “Belgorod State Agricultural University named after V. Gorin” is certified by the Sanitary and Epidemiological Service to * Corresponding author: [email protected] work with the sources of ionizing radiation (Toshiba D124 X-ray tube, DIG-360 X-ray) and provides conditions corresponding to the state sanitary and epidemiological standards under Sanitary Regulations 2.6.2612-10 “Basic Sanitary Rules for Radiation Safety”. The roentgenography was carried out under the following conditions: current strength – 320 milliamps, tube voltage – 45 kilovolts, focal range – 105cm [2], intensity – 65 kilovolts. The roentgenography was performed in the anteroposterior view. The lumbar of the bird was placed in the center of the X-ray film packet, and the body axis was sprawled along the film. Since making a quality image requires complete immobility of the patient, the manipulations were carried out by two professional veterinarians. Before the diagnostic procedures, the fowl with no detected abnormalities was weighed on the electronic scales, which allowed for the precise calculation of the radiopaque amount. To enhance the X-ray absorption and visibility of blood vessels, visceral cavities and tissues, 300 mg of iodine omnipaque were used. The omnipaque was injected via IV push, 2 ml per 1 kg. The sonography was carried out on the SIUI 5300 Apogee ultrasound system equipped with four transducers: C6LC microconvex transducer, C3LC macroconvex transducer, P5FC sector transducer and L8LC linear transducer; the ultrasound transducer frequency equaled 4-5MHz. The ultrasound propagation media included bones, parenchymal organs, soft tissues and gas characterized by different acoustic impedance and echogenicity. Technically, the examination was carried out on the patients laid down on the backbone. While being scanned, the cockerels were kept relatively immobile, their indumentum having been partly removed. Prior to the examination, their skin was surface-treated with ethanol and the ultrasound gel was applied. When imaging, the examined testicle was placed in the focal zone. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). BIO Web of Conferences 30, 06005 (2021) https://doi.org/10.1051/bioconf/20213006005 ILS 2020