3 Results and discussion According to different sources, surgeons have faced various complications doing orchiectomy in cockerels. The serious ones resulting in death of patients included hemorrhage due to major arteries and veins lesions as well as lesions in adjacent organs. Thus, orchiectomy in cockerels may result in lesion of the caudal air sac, the seventh rib fracture and development of the subcutaneous emphysema in the postoperative period [3]. The procedure allowed to study and describe the algorithm of the diagnostic roentgenography and sonography on cockerels of different ages. The applied methods allowed for the intravital painless diagnostics and for tracing the disposition of the testicles and the adjacent organs and major blood vessels as well as for the comparison of the obtained data and the data from alternative studies. At the preparatory stage of the experiment, we analyzed the works by N. E. Shalduga (2011), E. N. Nazarenko (2008) and E. N. Kuz’mina (2011) and, hence, discovered gaps in the presented materials: Surgical Caponizing of Cockerels by N. E. Shalduga lacks clarity in terms of differentiation of the topographic peculiarities of the cockerel reproductive organs based on age differences; Morphofunctional Peculiarities of Reproductive Organs Among Hisex Brown Cockerels in the PostIncubation Ontogenesis by E. N. Kuz’mina and Skeletopy of Cockerel Testicles and Major Blood Vessels by E. Y. Nazarenko and V. V. Degtjarjov focuses exclusively on the reproductive organs of Hisex Brown cockerels and do not contain any data on the anatomical peculiarities of D 959 cockerels. The results of the intravital methods of diagnostics confirmed the data provided in the abovementioned studies. The testicles of 60-day-old cockerels are imaged on both sides of the spinal column adjacent to the cephalic renal lobe and approaching the sixth rib on the frontal side. The testicles are adjacent to the posterior vena cava, most likely, due to short mesentery. At the rear and lateral sides, the testicles are partly covered by intestine loops. In the process of spermatogenesis, as reproductive organs grow, the testicles are adjacent to the walls of the major vessels: the ventral side is adjacent to the caudal vena cava and the dorsal side is adjacent to the thoracoabdominal aorta. The tail of the testicle is caudad to central or rear third of the cephalic renal lobe and may reach the external iliac vein [1,2,3]. 90-day-old D 959 cockerels have testicles adjacent to the frontal renal lobe and thoracic vertebral body; they reach the fifth rib in the cephalic position and are caudad to the lumbar vertebrae. At the rear and lateral sides, the testicles are partly covered by intestine loops. According to E. N. Kuz’mina [4], in puberty cockerel testicles are mostly placed asymmetrically and, as a rule, are slightly ectopic to the right side. The left testicle is adjacent to the thoracic vertebral body in the cephalic position and is caudad to the segments of the lumbar vertebrae whereas the right testicle has only its tail adjacent to them. In relation to the spinal column, both testicles take askew position; their tails are closely positioned at the base of the lumbar vertebrae, its second segment, though there are variations showing ectopy towards the first and the third segments. The cephalic boundary of the testicles is either the fourth intercostal space or the cephalic edge of the fifth or the sixth rib. The caudal boundary of the testicles is at the medial margin of the iliac bone (at the bottom of the aitchbone body) and the second segment of the lumbar vertebrae. The roentgenograms of 120-day-old D 959 cockerels show well-developed testicular arteries and veins. The testicles are much bigger in size in comparison to the three-month-old cockerels. They are placed between the fourth intercostal space and the second segment of the lumbar vertebrae. At the rear and lateral sides, the testicles are partly covered by intestine loops. The sonography in 60-day-old cockerels proved to be ineffective, apparently due to yet underdeveloped testicles and the lay-over effect. The testicles of 90-day-old cockerels were shaped as an elongated oval, had well-defined and smooth boundaries with homogeneous echotexture and were characterized by hyperechogenicity, which was defined as a norm. The length of the right testicle amounted to 0.9 cm and the width – to 0.39 cm. The length of the left testicle equaled 1.3 cm and its width equaled 0.5 cm. Neither the epididymides nor neoplasms were imaged. The testicles of 120-day-old cockerels were beanlike, had well-defined and smooth boundaries and were characterized by homogeneous echotexture and hyperechogenicity, which was defined as a norm. The length of the right testicle amounted to 4.5 cm and the width amounted to 2.6 cm. The length of the left testicle equaled 4.61 cm and its width equaled 2.9 cm. Neither the epididymides nor neoplasms were imaged. The abovementioned non-invasive examination methods were used as the complementary methods that together allowed to study the topography of the cockerel testicles, define their sizes, plan the surgery procedure and evaluate the possibility of various further complications. Roentgenographic examination and diagnostic sonography are two simple and timesaving procedures which allowed to define the optimal age for orchiectomy in cockerels. Defining the optimal age for orchiectomy in cockerels was based the following criteria: surgical procedure with minimal time input and minimal postoperative complications. The obtained data showed that the optimal age for orchiectomy in cockerels is 60 days. In the course of the research, we arrived to the following conclusions: Testicles of 120-day-old cockerels have welldeveloped blood vessels and significant size, which may lead to a failed surgery and post-operative complications. Roentgenographic examination and diagnostic sonography allow to carry out intervital diagnostic examination. Roentgenographic examination and diagnostic sonography put the surgeon into control of the whole process of the surgical procedure and its outcome. As the transducer can only be applied to a limited area of the body surface and the indumentum hinders the process, sonography cannot be considered fully efficient. 2 BIO Web of Conferences 30, 06005 (2021) https://doi.org/10.1051/bioconf/20213006005 ILS 2020
If carried out as a complex examination, roentgenography and diagnostic sonography are complementary to each other. Diagnostic sonography offers little information if the examination is carried out on cockerels under the age of 90 days of post-incubation ontogenesis. The above instrumental methods are not sufficient for the thorough study of the species peculiarities in terms of topography and perfusion in D 959 cockerels. 4 Conclusion The diagnostic imaging is an indispensable part of the complex examination which allows for the formation of a complete picture of the surgical procedure complexity within a short period of time and helps a surgeon to plan a surgical procedure, predict the risks and decide if the surgery has to be carried out urgently and, hence, have a critical insight into the procedure and perform the surgery adequately. The market of veterinary equipment has a wide range of producers, models and software for roentgenography and diagnostic sonography. However, the quality of the equipment does not always guarantee acquisition of the precise data. Following the necessary stages of the examination, experience and knowledge of basic animal and bird anatomy greatly contribute to building the complete picture. The current study can be helpful for practicing veterinarians and students keen on studying common modern methods of diagnostics. Orchiectomy in cockerels allows producing meat in which the amino acid balance corresponds to that of a perfectly balanced animal protein that contains the optimal ratio of all the amino acids [5]; further research on this issue appears to be of great relevance due to the rapid development of the research and education centers engaged in quality and healthy food production. References 1. G. M. Udovin, International Veterinary Anatomy Nomenclature in Latin and Russian (MVA, Moscow, 1979). 2. E. N. Nazarenko, and V. V. Degtjarjov, The Bulletin of the Orenburg State Agricultural University, 4, 111-113 (2008). 3. N. E. Shalduga, Surgical Caponizing of Cockerels. Disseratation, extended abstract, (Kharkov Veterinary University, Kharkov, 1952). 4. E. N. Kuz’mina, Morphofunctional Peculiarities of Reproductive Organs Among Hisex Brown Cockerels in the Post-Incubation Ontogenesis (Dissertation, Orenburg, 2009). 5. T. M. Giro, S. A. Zlobina, and S. I. Khvylja, Poultry and Poultry Products, 6, 64-66 (2014). 3 BIO Web of Conferences 30, 06005 (2021) https://doi.org/10.1051/bioconf/20213006005 ILS 2020
The productivity of boars when introduced to the feed sprouted grains Aleksandr Kovrigin 1* , Grigorii Pokhodnya1 , Olga Yastrebova1 , and Yuriy Breslavets1 1 Belgorod State Agricultural Univerisity named after V.Gorin. vil. Mayskiy, Russia Abstract. Recipes were developed for compound feeds for boars with replacement of 5% and 10% of natural barley grain with sprouted grain. The use of experimental feeds allowed to increase the volume of boar ejaculate-by 25.5% and 31.9%; sperm concentration-by 18.4 and 20.1%; sperm survival-by 7.7 and 9.2 hours; sperm resistance - by 27.0 and 27.8%. The fertilization rate of sows increased by 3.0 and 6.3%, multiplicity-by 3.1 and 4.2%, large-scale fertility remained at the control level. The cost of compound feed increases with the introduction of sprouted grain, but this increase is economically justified, since the productivity of animals increases. The cost of a dose of sperm from boars is reduced by 2.8 and 3.2 rubles, the number of farrowed sows increases in the experimental groups by 51.8 and 71.4%, respectively, and the cost of one Piglet is lower by 12.4 - 17.5 rubles or 8.6 - 12.6%.. 1 Introduction For intensive pig farming, it is important to use boars efficiently and increase their reproductive functions. Attempts were made to improve the productive qualities of animals and poultry by various methods: optimizing individual parameters of the microclimate [1], using stimulating drugs and adaptogens, and influencing the sexual sphere with hormonal and other means [2-5]. However, these techniques are not sufficiently implemented in practice due to the high cost of drugs and the complexity of their use in production conditions.in our opinion, it is possible to stimulate the reproductive function of boars much cheaper with feed [6,7]. It is known that sperm production and secretion of the additional sex glands of boars require a large amount of essential amino acids, fatty acids, and other important biological components as a structural material. In addition, studies by a number of authors [8-10] show that each ejaculation of a boar is accompanied by a significant loss of vitamins a, C and others by the body. Therefore, these important components should be included in the compound feed for boars. However, in the conditions of market relations, prices for compound feeds and, especially for high-energy additives, protein and vitamin concentrates, premixes, dry concentrate and other components have sharply increased. Therefore, the method of sprouting grain components can significantly increase their usefulness and at the same time reduce the share of expensive components (premixes). The use of sprouted grain in compound feeds is a simple, affordable and inexpensive way to increase the vitamin value of diets. In this case, the grain is converted into a dietary feed that exceeds the original one in terms * Corresponding author: : [email protected] of proteins, essential amino acids, macro-and microelements, vitamins A, C, E, and group B [11]. A number of chemical compounds pass from complex forms to simpler and easily digestible ones. So, starch decomposes to simple Sugars, proteins to amino acids, fats to fatty acids. The amount of soluble nitrogenous compounds increases, and high - energy compoundsphospholipids-are also formed [12]. 2 Materials and Methods To study the effectiveness of using sprouted barley grain in the diets of boars-producers, we conducted a scientific and economic experiment at the Gorin collective farm in the Belgorod region, where three groups of Welsh boars aged 2-3 years were selected according to the principle of analogues. The conditions of detention and sexual use for boars of all three groups were the same. During the preparatory period, which lasted 40 days, boars of all experimental groups were fed standard K-57-2 compound feed of 4 kg per day per head. During the experimental period, the boars of the first control group received the same K-57-2 compound feed, the animals of the experimental groups were fed " experimental compound feed, which included 5 (second) and 10% (third group) of sprouted barley grain instead of natural. The boars were fed twice a day according to the VIZ standards, and one head was kept individually in the machine. The improved formula of compound feeds for boarsproducers, used in scientific and economic experience, is presented in Table 1. It should be noted that in experimental mixed feeds, in comparison with the standard, the number of feed units and exchange energy decreased by 1.9-2.9 and 1.7-3.5%, © 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, 06006 (2021) https://doi.org/10.1051/bioconf/20213006006 ILS 2020
respectively. The reason for this is that during the germination of grain, the concentration of dry matter decreases due to the partial loss of organic substances that went to the respiration processes. Therefore, the dry matter content in mixed feeds, which included sprouted barley, decreased by 1.4-3.1%. The concentration of raw and digestible protein did not change significantly and was at the level of the control variant. The amount of raw fiber in experimental feed is slightly reduced, but not to such an extent as to focus attention on it. The amount of macronutrients of calcium and phosphorus remained at the same level and did not depend on the feed recipe. The content of some trace elements (iron, copper, zinc and manganese) in the experimental versions of compound feeds decreased insignificantly, while cobalt and iodine increased. When recalculating these mineral substances to absolutely dry feed substance, there is an increase in their compound feed. Since the method of germination significantly affects the concentration of biologically active substances in the grain, the experimental batches of compound feed showed an increase in vitamins B1, B2 and E: by 1.4-4.5%. The carotene content increased by 0.6-1.2%. Table 1. Сomposition and nutrition of compound feeds for boars-producers, % Ingredients Control To the-57-2 Experimental №1 №2 % input of sprouted grain 5 10 Corn 22,2 22,2 22,2 Barley 14 9/5٭ 4/10٭ Oats 16 16 16 Wheat bran 9 9 9 Sunflower meal 17 17 17 Fish meal 9 9 9 Bone meal 1 1 1 Herbal flour 10 10 10 Desulfurized phosphate 0,4 0,4 0,4 Salt 0,4 0,4 0,4 Premix P-57-1 1 1 1 1 kg contains: Feed units 1,05 1,03 1,02 Exchange energy, MJ 11,68 11,48 11,28 Dry matter, g 860,0 848,0 834,0 Digestible protein, g 188,1 187,2 186,2 Crude fiber, g 79,1 79,0 78,8 Calcium, g 12,3 12,3 12,3 Phosphorus, g 9,9 9,9 9,9 Iron, g 168,2 167,0 165,8 Crude protein, g 222,1 220,9 219,6 Copper, mg 18,8 18,7 18,7 Zinc, mg 72,1 71,8 71,5 Manganese, mg 40,2 40,1 40,0 Cobalt, mg 1,69 1,70 1,72 Iodine, mg 0,67 0,77 0,89 Carotene, mg 16,6 16,7 16,8 Vitamin B1, mg 4,1 4,3 4,3 Vitamin B2, mg 4,2 4,3 4,4 2 BIO Web of Conferences 30, 06006 (2021) https://doi.org/10.1051/bioconf/20213006006 ILS 2020
3 Results and discussion The introduction of sprouted barley grains instead of natural ones into K-57-2 feed increased the sperm production of boars. (Table 2 and Table 3). At 5% replacement (the second group), the volume of ejaculate increased by 25.5% compared to the control group, and at 10% replacement (the third group) - by 31.9%. The concentration of sperm in the second and third groups increased by 18.4 and 20.1%, respectively, compared to the preparatory period. When comparing the experimental groups of boars with the control variant for this indicator in the experimental period of the experiment, the increase in the concentration of sperm in the second and third groups was, respectively, 44.5 and 50.6 million / ml (or by 17.5 and 19.9%). The increase in the first two indicators of sperm production (volume and concentration) from feeding the experimental compound feed to boars naturally increased the total number of sperm in the ejaculate. When comparing this indicator between the periods of the experiment, the movement of sperm in the second and third groups was, respectively, 50.0 and 59.8% (p <0.01), and in the experimental period the increase in these groups with the control was at the level of 24.39 and 29 , 90 billion (or 47.4 and 58.2%). Having studied the motility of sperm, we found that this indicator was quite high in all groups. At the same time, intergroup differences were insignificant and practically did not depend on the fed compound feed. Along with motility, an important indicator of the quality of boar sperm is the survival (vitality) of sperm outside the body. As a result of the control over this test, it was found that the survival rate of sperm increases significantly with the use of mixed feed with germinated barley grain in the feeding of boars. With 5 and 10% introduction of such grain, the survival rate of spermatozoa in the second and third groups increased in comparison with the preparatory period by 7.7 and 9.2 hours, respectively. Of great importance for the qualitative characteristics of sperm is its indicator - resistance. This test shows the resistance of sperm cells to the action of 1% sodium chloride solution. This method is based on the action of this salt on the protected lipoprotein membrane of spermatozoa, which protects them from possible harmful effects of the environment. The indicator of resistance is very important, since it is associated with the vitality of sperm, reflects their biological value and fertilizing ability. In our studies, sperm resistance increased with an increase in the dose of germinated grain in the feed. So, in the second and third groups, this indicator increased, compared with the control variant, by 27.0 and 27.8%, respectively. Thus, replacing a part of the natural barley grain in the K-57-2 compound feed with germinated ones by 5 and 10% significantly increased the volume of ejaculate, the total number of sperm and their survival rate. This replacement also contributed to an increase in sperm concentration, motility and sperm resistance. Moreover, the best indicators for sperm production were obtained with a 10% inclusion of germinated grain. The efficiency of insemination of sows with freshly harvested and frozen semen of experimental boars is presented in Tables 4 and Tables 5. TABLE 2. Indicators of boar sperm production Indicators Groups preparation period І ІІ ІІІ control K-57-2 The volume of ejaculate, (ML) 201.5 ± 3.85 200.0 ± 14.64 200.3 ± 3.93 Concentration sperm cells, mln / ml 253.7 ± 27.0 252.6 ± 1 8.0 254.0 ± 10.4 The total number of spermatozoa in the ejaculate are , bn 5 1 . 1 2 ± 1 . 1 3 50.52 ± 1 .31 50.88 ± 1 . 1 0 Sperm motility, points 8.00 ± 0.006 7.93 ± 0.006 7.96 ± 0.006 Sperm survival, h 66.2 ± 1 . 1 3 64.8 ± 1.41 65.0 ± 1.20 Sperm resistance 950 ± 62.9 943 ± 91.3 956 ± 89.6 Table 3. Indicators of boar sperm production Indicators Groups trial period I (К-57-2) ІІ ІІІ % input of germinated grain 0 five ten The volume of ejaculate, (ML) 202.0 ± 4.3 1 253.5 ± 5.45 ** 266.5 ± 6.10 ** Concentration sperm cells, mln / ml 254.5 ± 1 3 .4 299.0 ± 42.8 305.1 ± 33.0 The total number of spermatozoa in the ejaculate are , bn 5 1.41 ± 1.29 75.80 ± 1.88 ** 81.31 ± 1.73 ** Sperm motility, points 8.03 ± 0.003 8.00 ± 0.003 8.06 ± 0.003 Sperm survival, h 65.7 ± 0.95 72.5 ± 0.75 *** 74.2 ± 1.07 *** Sperm resistance 968 ± 90.9 123.1 ± 87.2 1237 ± 55.4 Data from Table 6 show that the fertilization rate of sows in the experimental groups increased by 3.0 and 6.3%, multiplicity-by 3.1 and 4.2%, and large-scale fertility remained at the level of the control variant. When using frozen sperm for insemination of sows, there is a tendency to increase in comparison with the control of the above-mentioned indicators, except for large-fruiting (Table 6,7). Thus, the fertilization rate of Queens increased by 6.6 and 10.0%, and multiple pregnancies-by 7.2 and 8.0%. Large-fruited remained at the control level. 3 BIO Web of Conferences 30, 06006 (2021) https://doi.org/10.1051/bioconf/20213006006 ILS 2020
Data from Table 8 show that with the same consumption of compound feed, its cost in the second and third groups was higher compared to the control by 12.4 and 24.8 rubles. However, this difference was completely covered by receiving additional spermodoses from boars of the second and third groups due to an increase in the volume of ejaculate and the concentration of sperm in it. The cost spermatozoa decreased in comparison with control PA 2,8 and 3,2 RUB. Using this sperm to inseminate sows also had a positive effect. The number of farrowed sows increased in the experimental groups by 51.8 and 71.4%, respectively. The above mentioned caused an increase in the cost of maintaining sows and piglets in the experimental groups, but the cost of one pig became lower than PA .12, 4-17.5 rubles or 8.6-12.6%. At the same time, it is more costeffective to use compound feed for boars with a 10% inclusion of sprouted barley. This feeding allows producers to improve quantitative and qualitative characteristics of sperm, increase the number of sperm doses and reproductive qualities of sows from using semen of Guinea pig boars for insemination. The effectiveness of the use of experimental compound feed for boars is reflected in Table 8 Table 4. Reproductive functions of sows when they are inseminated with fresh sperm Indicators Groups preparation period І ІІ ІІІ control K-57-2 Sows inseminated thirty thirty thirty of them farrowed 25 24 24 in % 83.3 80.0 80.0 Plurality, heads 9.52 ± 0.2 9.50 ± 0.2 9.60 ± 0.2 Large-fruited, kg 1.22 ± 0.01 1.25 ± 0.01 1.21 ± 0.02 Table 5. Reproductive Functions Of Sows When They Are Inseminated With Fresh Sperm. Indicators Groups preparation period I (К-57-2) ІІ ІІІ % input of germinated grain 0 five ten Sows inseminated 50 60 60 of them farrowed 41 51 53 in % 82.0 85.0 88.3 Plurality, heads 9.60 ± 0.1 10.0 ± 0.2 9.90 ± 0.1 Large-fruited, kg 1.25 ± 0.01 1.23 ± 0.02 1.24 ± 0.01 Table 6. Reproductive functions of sows during insemination with frozen sperm Indicators Groups preparation period І ІІ ІІІ control K-57-2 Sows inseminated thirty thirty thirty of them farrowed nineteen 20 20 in % 63.3 66.7 66.7 Plurality, heads 8.72 8.83 8.79 Large-fruited, kg 1.35 1.34 1.35 Table 7. Reproductive functions of sows during insemination with frozen sperm Indicators Groups trial period I (К57-2) ІІ ІІІ % input of germinated grain 0 five ten Sows inseminated thirty thirty thirty of them farrowed 20 22 23 in % 66.7 73.3 76.7 Plurality, heads 8.80 9.50 9.43 Large-fruited, kg 1.37 1.35 1.36 Table 8. The effectiveness of the use of experimental compound feeds in the diets of boars (per head for the experimental period) Indicators Groups І ІІ ІІІ Combined feed consumed, c 2.48 2.48 2.48 The cost of the consumed compound feed, rub. 744.0 756.4 768.8 Boar maintenance costs, rub. 1240.0 1252.4 1264.8 Sperm dose received 138 202 218 Sperm dose cost, rub. 9.0 6.2 5.8 Sows inseminated 69 101 109 Of them farrowed, goal. 56 85 96 Multiple pregnancy, goal. 9.6 10.0 9.9 Received piglets, goal 537 850 950 Costs for the maintenance of sows for 120 days. rub. 82800 121200 130800 Total costs for piglets received, rubles 84042.0 122454, 132064.4 Cost of one pig at birth, rub. 156.5 144.1 139.0 Difference with control, rub. - -12.4 -17.5 4 BIO Web of Conferences 30, 06006 (2021) https://doi.org/10.1051/bioconf/20213006006 ILS 2020
4 Conclusion The introduction of sprouted grain in the diets of boars in the amount of 5 and 10% by weight showed a positive effect on their productivity. Thus, the volume of ejaculate increased by 25.5% compared to the control group, and at 10% - POI (the third group) - by 31.9%. The concentration of sperm in the second and third groups increased by 18.4 and 20.1%, respectively, compared to the preparatory period. The survival rate of spermatozoa in the second and third groups increased by 7.7 and 9.2 hours, respectively, in comparison with the preparatory period. Sperm resistance in the second and third groups increased by 27.0 and 27.8%, respectively, compared to the control variant. The fertilization rate of sows in the experimental groups increased by 3.0 and 6.3%, multiplicity-by 3.1 and 4.2%, large-scale fertility remained at the level of the control variant. The cost of compound feed increases with the introduction of sprouted grain, but this increase is justified, since the productivity of animals increases. The cost of a dose of sperm from boars is reduced by 2.8 and 3.2 rubles, the number of farrowed sows increases in the experimental groups by 51.8 and 71.4%, respectively, and the cost of one Piglet is lower by 12.4 - 17.5 rubles or 8.6 - 12.6%. Thus, the introduction of sprouted barley grain in the amount of 5% and 10% in the diet of boars is economically justified. References 1. S.A. Eriomin, and O.N. Durykhina, “The use of computers to control the microclimate in poultry farms,” Proceedings of the International student scientific conference, Belgorod: Publishing house of BelSAU, pp.80-81, April 2008. 2. G.S. Pokhodnya, A.V. Kovrigin, and A.M. Mamenko “Innovative technologies of using the unconventional feed in the production of agricultural products,” Problems of Zooengineering and Veterinary Medicine, Kharkiv, no. 31-1, pp.164- 166, 2015. 3. N.N. Shvetsov, G.S. Pokhodnya, and S.P. Salamakhin “New feeds with extruded grains,” Stock Farming of Russia, no. 10, pp. 43-44, 2009. 4. O.N. Yastrebova, Feeding of farm animals: Textbook for students of SVE specialty 36.02.02 Animal Science, Belgorod: Belgorod SAU, 2016. 5. V.A. Panarina, and O.N. Yastrebova, “Cultivation of fish and plants with aquaponic technology,” Gorin readings. Science of young – for innovative development of agriculture. Materials of the international student scientific conference, Belgorod: Belgorod SAU, pp. 126-127, 2019. 6. A.V. Kovrigin, “Automated technology of manufacture of ecologically clean products in crop production and aquaculture in controlled conditions of premises,” Innovations in Agricultural Complex: Problems and Perspectives, Belgorod: Belgorod SAU, no. 4 (12), pp. 124-129, 2016. 7. A.V. Kovrigin, A.P. Khokhlova, and N.A. Maslova “Study of the efficiency of operation of an automated aquaponic installation depending on the modes of its operation,” Bulletin of KrasGAU, pp. 90-96, no. 11, 2015. 8. A.V. Kovrigin, Applied computer programs in breeding and feeding. Textbook, public house of Belgorod SAU, 2010. 9. A.V. Kovrigin, “Some aspects of the development of innovative technologies of pork production in medium and small agricultural enterprises of Russia,” Collection of scientific works of the scientific school of professor G.S. Pokhodnya, Issue no. 4, Belgorod: publishing house of BelSAU, pp. 48-49, 2011. 10. G.S. Pokhodnya, E.G. Fedorchuk, A.I. Grishin, and R.A. Strelnikov “Suspension of chlorella in rations of sows,” Vestnik of Kursk State Agricultural Academy. Kursk: Public house of Kursk State Agricultural Academy, no. 4, pp. 47-48, 2012. 11. G.S. Pokhodnya, P.P. Kornienko, and A.V. Kovrigin, Increase of the reproductive capacity of pigs. Belgorod: Public house of Belgorod SAU, 2013. G.S. Pokhodnya, N.I. Bogdanov, E.G. Fedorchuk, A.I Grishin., N.P. Dudina, and Yu.P. Breslavets, Recommendations for the use of chlorella suspension in the diets of pigs. Belgorod, 2012 5 BIO Web of Conferences 30, 06006 (2021) https://doi.org/10.1051/bioconf/20213006006 ILS 2020
Bovhyaluronidaze azoximer as an antimicrobial alternative in companion animals Anna Nazarova 1* , Boris Semenov 1 , Anna Kozitcyna 1 , Veronika Guseva1 , Tatiana Kuznetsova1 , and Vladimir Videnin 1 1Saint-Petersburg State University of Veterinary Medicine, Saint Petersburg, Russia Abstract. Antimicrobial resistance formation in various bacteria is a serious biosafety threat. In this regard, the important issue in medical and veterinary fields is not only the rational use of antibacterial drugs, but also the search for ways to significantly reduce the need for antibiotics. Three studies designed to research into these challenges are described. The results indicated that (1) the bacteriuria presences in 62.4 +/- 4.1% of urine samples obtained from companion animals (the microflora count in urine samples is estimated from '++' to 'Entire Field-of-View'); (2) 16.3 +/- 4.0% of samples from companion animals showed bacteria with low antibiotics sensitivity, and 8.1 +/- 3.0% of samples contained bacteria resistant to 30 antibiotics included in the susceptibility test; (3) the Bovhyaluronidaze azoximer reduce antimicrobial therapy duration statistically significant (p=0.0089). The studies were conducted at the facilities of St. Petersburg State University of Veterinary Medicine and Bars veterinary clinic chain in St. Petersburg, Russia. A randomized clinical trial of Bovhyaluronidaze azoximer effect on course of disease and treatment duration of acute and chronic prostatitis and urocystitis, including complicated by urolithiasis, was conducted in 54 dogs. Animals were randomly assigned to intervention (n=34) and control (n=20) groups. Animals of both groups received the antimicrobial therapy. Interventional group patients additionally received Bovhyaluronidaze azoximer in the form of rectal suppositories. The antimicrobial therapy mean duration in intervention group animals took 14.88 +/- 4.98 days, in control group animals — 18.65 +/- 5.40 days. The study findings imply that bacteriuria is widely prevalent in companion animals and antimicrobial resistance is increasing issue in veterinary medicine. Bovhyaluronidaze azoximer contributes to microcirculation restore and increases the bioavailability of antibacterial drugs at the site of infection. This makes its relevant for use in the complex treatment for septic and inflammatory processes. This allows to reduce the antimicrobial course duration (and completely decline antibiotic therapy in some cases) and prevent the formation of chronic inflammation and infection sites. 1 Introduction Currently, due to the fast rate of antimicrobial resistance widespread in various bacteria, an important biosafety issue in both medical and veterinary fields is not only the rational use of antimicrobial agents, but also the search for medications that can significantly reduce the need for antibiotics. An important component of antimicrobial resistance problem is the formation of bacterial biofilms. Bacteria living in a biofilm can be protected from both antimicrobial agents and immune system [1]. As an alternative to antibiotics, research is being carried out on the possibility of using pharmaceutical preparations based on silver such as Silver Chloride Gel [2] and Argentum Quartz (R) [3], probiotics such as Lactobacillus plantarum [4], Curcumin oile [5] and new types of bandages, for example the Lawsonia Inermisgelatin-starch nano-fibrous bandage [6]. However, these methods are only for topical use and do not have a systemic effect, so they does not act upon the site of chronic infection (which often forms after the biofilm formation), and also does not affect the immune system. * Corresponding author: [email protected] Urinary tract infections are the common reason for the antimicrobial use in veterinary medicine, which promotes antimicrobial resistance in urological patients [7, 8]. The Department of Obstetrics and Operative Surgery of the St. Petersburg State University of Veterinary Medicine underwent studies of Bovhyaluronidaze azoximer (lat.: Bovhyaluronidazum azoximerum), a stabilized form of enzyme hyaluronidase (hyaluronidase conjugate with a high-molecular derivative of poly-1,4- ethylene piperazine N oxide). Development and production — NPO “Petrovax Pharm”, Russia. Studies of Bovhyaluronidaze azoximer use are carried out in the following areas: • prevention of postoperative complications in urological patients who underwent bladder and urethra surgery; • treatment of animals with purulent-inflammatory processes in various sites; • treatment of animals with chronic urocystitis, including complicated by urolithiasis, as well as with chronic diseases of the prostatic gland. © 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, 06007 (2021) https://doi.org/10.1051/bioconf/20213006007 ILS 2020
2 Experimental The studies were conducted at the facilities of Bars veterinary clinic chain in the city of St. Petersburg, Russia, and St. Petersburg State University of Veterinary Medicine. Laboratory tests, including urinalysis, urine bacterial culture and antimicrobial susceptibility tests, were performed in the Bars-Diagnostics laboratory, St. Petersburg. To assess the prevalence rate of bacteriuria the microflora count in urine samples from 141 cats was evaluated. To assess the prevalence of antimicrobial resistance, the results of a disc-diffusion antibiotic sensitivity test were analyzed in 86 samples (57 dogs and 29 cats). 30 antibacterial agents were included in the tests: Amoxicillin, Ampicillin, Azithromycin, Aztreonam, Bacitracin, Cefazolin, Cefotaxime, Ceftriaxone, Clarithromycin, Clindamycin, Doxycycline, Enrofloxacin, Erythromycin, Furadonin, Furagin, Furazolidone, Imipenem, Levofloxacin, Lincomycin, Lomefloxacin, Nalidixic acid, Norfloxacin, Novobiocin, Ofloxacin, Oxacillin, Polymyxin B, Tetracycline, Trimethoprim / Sulfamethoxazole, Tylosin, Vancomycin. Study of Bovhyaluronidaze azoximer effect on the course of disease and treatment duration of acute and chronic prostatitis and urocystitis, including complicated by urolithiasis, was conducted in 54 animals. In this randomized clinical trial 54 dogs (Canis lupus familiaris) with urogenital system diseases were randomly assigned to intervention (n=34) and control (n=20) groups. The intervention group included 22 males and 12 females. The control group included 15 males and 5 females. The mean age of the dogs in the intervention group was 6.24 ± 2.83 years, in the control group — 6.80 ± 3.69 years. The mean weight of the dogs was 7.62 ± 8.72 kg and 12.09 ± 9.71 kg in the intervention and control groups respectively (Standard Deviation (SD) is indicated). Prostatitis was diagnosed in 19 dogs (10 dogs in the intervention group and 9 dogs in the control group). Bacterial urocystitis was diagnosed in 24 dogs (16 dogs in the intervention group and 8 dogs in the control group). Bacterial urocystitis complicated by urolithiasis was diagnosed in 9 dogs (7 dogs in the intervention group and 2 dogs in the control group). One dog in the experimental group had a penile trauma and 1 dog in the control group had acute urinary retention. Clinical examination of all animals was carried out in accordance with GOST R 58090-2018 “Clinical examination of unproductive animals” [9]. The diagnosis was established on the basis of anamnesis, physical examination, the results of the urinalysis and visual diagnostic data (ultrasound and x-ray studies). The animals of both groups receiver antibiotic therapy in accordance with generally accepted recommendations for the use of drugs and in accordance with the results of the antibiotic susceptibility test (in those cases when these tests were performed). Patients of the interventional group additionally received Bovhyaluronidaze azoximer in the form of rectal suppositories. The effectiveness of antibiotic therapy was assessed by the presence of microflora in the controlled urine samples. We performed the statistical analysis in Microsoft Excel 2016 and BioStat, AnalystSoft Inc., version 7. The statistical significance (alpha level) adopted in this study is 5% (p=0.05). To compare the treatment duration in the interventional and control groups the Mann-Whitney U Test was used. If the significance level p obtained by applying the criterion was less than 0.05, the differences between the groups were recognized as statistically significant. 3 Results and discussion Bacteriuria prevalence Table 1 presents the results of a statistical analysis of 141 urine samples obtained from animals admitted with symptoms of urinary tract distress. Only in 37.6 ± 4.1% of the samples were detected no microflora or the count was insignificant ('+') (hereinafter, Standard Error of Mean (SEM) is indicated). In 62.4 ± 4.1% of the samples, the microflora count was estimated from '++' to 'Entire Fieldof-View'. Thus, it is established that in most cases in animals with urinary tract diseases antimicrobial therapy should be part of a complex therapy. However, errors such as unreasonable prescription of antibiotics for the prevention of bacterial infections, improper selection of dose or administration frequency and course duration can reduce the effectiveness of antimicrobial therapy, as well as lead to a variety of side effects, such as the disease transition into a chronic form and the formation of antimicrobial resistance bacteria strains. Antimicrobial resistance prevalence In veterinary practice, antibiotic susceptibility test is not carried out routinely during the initial treatment in the clinic. If a bacterial cause is suspected, antimicrobial agent is empirically prescribed. Often, with the ineffectiveness of the first antibacterial agent, an antibiotic change is also carried out empirically, without an antibiotic susceptibility test. Table1. Microflora in the urinalysis samples The count of microflora a Percentage of the urinalysis samples (%) Percentage ± SEM – 1.4 ± 1.0 + 36.2 ± 4.1 ++ 19.1 ± 3.3 +++ 5.7 ± 2.0 ++++ 2.8 ± 1.4 Entire Field-ofView 34.8 ± 4.0 As a result, by the time the animal owners agreeing to conduct antibiotic susceptibility test, there is often an association of microorganisms in the infection site, and/or microorganisms that have developed resistance to a large number of antibiotics. Eighty sex results of a disc-diffusion antibiotic sensitivity tests were analyzed. The animals mean age from which the samples were taken was 6.6 ± 4.0 years (± SD is indicated). 2 BIO Web of Conferences 30, 06007 (2021) https://doi.org/10.1051/bioconf/20213006007 ILS 2020
Of these, 44 samples (51.2%) were the urine samples, 30 samples (34.9%) were the materials from the ears, 6 samples (7.0%) were the materials from the eyes, and 3 samples (3.5% each) were the materials from the larynx and skin. A culture of Staphylococcus aureus was found in 97.7 ± 1.6% (hereinafter ± SEM is indicated) of the samples, and Escherichia coli in 37.2 ± 5.2% of the samples. In 44.2 ± 5.4% of the samples, an association of microorganisms was found. Oxacillin resistance was established in microorganisms from 66.3 ± 5.1% of samples. There were detected low antibiotic sensitivity for bacteria from 16.3 ± 4.0% of the samples (for 30 antibacterial agents included in the tests), and bacteria from 8.1 ± 3.0% of the samples had antibiotic resistance to all 30 antibiotics included in the tests (all 30 antimicrobial agents were ineffective). In the 44 urine samples culture of Staphylococcus aureus was found in 95.5 ± 3.2% (hereinafter ± SEM is indicated) of the urine samples, and Escherichia coli in 52.3 ± 7.6% of the urine samples. In 52.3 ± 7.6% of the urine samples, an association of microorganisms was found. Oxacillin resistance was established in microorganisms from 75.0 ± 6.6% of urine samples. There were detected low antibiotic sensitivity for bacteria from 18.2 ± 5.9% of the samples (for 30 antibacterial agents included in the tests), and bacteria from 15.9 ± 5.6% of the urine samples had antibiotic resistance to all 30 antibiotics included in the discdiffusion antibiotic sensitivity tests (all 30 antimicrobial agents were ineffective). Therefore, it is established that urine samples bacterial culture possess higher antibacterial resistance in comparison with samples from other sites. Therapy of patients with antimicrobial resistant microflora requires not only the selection of antimicrobial agents and their use in an adequate dose and sufficient course duration, but also the bioavailability of the antibiotic in the inflammation site and prevention chronic inflammation and infection sites formation. Chronic urocystitis and prostatitis treatment duration reduction In the treatment of chronic urocystitis and prostatitis in intervention group animals the Bovhyaluronidaze azoximer in the form of suppositories was administered rectally in addition to antimicrobial therapy. For dogs of the intervention group weighing up to 10 kg, the Bovhyaluronidaze azoximer was administered at a dose of 750 IU per animal, for dogs weighing up to 30 kg the Bovhyaluronidaze azoximer was administered at a dose of 1,500 IU per animal, and for dogs weighing more than 30 kg — 3,000 IU Bovhyaluronidaze azoximer per animal. Bovhyaluronidaze azoximer suppositories were administered rectally q48h, 10 administrations, and then q72h, 10 administrations. One animal experienced bleeding after the drug administration (further use was discontinued). In 3 animals, dose-related side effects were observed in the form of lethargy and appetite loss. After dose reducing to 500 IU of Bovhyaluronidaze azoximer per animal, the side effects ceased. The mean antimicrobial therapy duration in animals of the intervention group was 14.88 ± 4.98 days (± SD is indicated). The mean antimicrobial therapy duration in animals of the control group was 18.65 ± 5.40 days (± SD is indicated). When comparing the results obtained in the intervention and control groups using the Mann-Whitney U Test, the p-value is 0.0089, which is much less than the alpha level adopted in our study (p=0.05) and allows us to conclude that the antimicrobial therapy length reduction in animals of the intervention group is statistically significant. A summary diagram of the length of treatment for 3 diseases is presented in Fig. 1. Mechanism of action of Bovhyaluronidaze azoximer on the course of inflammatory processes Urgent conditions such as acute or recurrent urinary retention, protracted chronic urinary retention caused by obstruction and/or urethral stenosis, injuries and ruptures of the urethra require surgical interventions such as cystocentesis, bladder catheterization, cystotomy, perineal urethrostomy in order to save animal’s life. In this regard, the goal is to minimize the complications that may be caused by such interventions. The most common complications of perineal urethrostomy in the short term are: hematomas, subcutaneous urine infiltration in the area of urine leakage between the urethra mucous membrane and the skin, repeated urethra obstruction with blood clots and/or due to edema of urethra mucous membrane and periurethral tissue, excoriation of the perineum and suture failure [10]. Fig. 1 Confidence intervals for 3 diseases in the intervention and controlgroups In long term, complications of perineal urethrostomy may include chronic dermatitis of the skin fold, the bladder atony, urinary incontinence, and urethral stricture [11]. Urethral strictures and urethrostomy overgrowth, according to different authors, range from 12% [12] to 35% [13]. Recurrent urinary tract infection is observed in more than 30% of cats undergoing perineal urethrostomy and in more than 60% of cats undergoing prepubic urethrostomy [14, 15]. Complications such as postoperative urinary tract infections are also observed 3 BIO Web of Conferences 30, 06007 (2021) https://doi.org/10.1051/bioconf/20213006007 ILS 2020
after cystotomy [16]. In this regard, antimicrobial agents are often used during postoperative period. Acute inflammatory process is accompanied by the edema formation. Edema disrupts adequate blood supply, reduces the bioavailability of antibacterial drugs, as a result tissue healing slows down, and chronic inflammation and infection site is created. The chronic inflammatory process that forms as a result of prolonged edema in 100% of cases is characterized by the formation of scar (granulation) tissue [17]. The degree of edema (including edema of periurethral tissues after perineal urethrostomy) significantly affects the development of postoperative complications. The chronic inflammatory process caused by prolonged edema leads to fibrotic changes in the periurethral tissues, increases the rigidity of the urethra and bladder, causing relapses of acute urinary retention. Also, the formation of a site of chronic infection, incomplete emptying of the bladder, urine stagnation and relapses of acute urinary retention lead, in turn, to the formation of antimicrobial resistant bacteria strains, including oxacillin-resistant strains. As a result, the following tasks are set in the course of inflammatory process treatment: -reduce swelling and tissue inflammation, -restore microcirculation, -ensure higher antibiotics bioavailability in the infectious site, -reduce the synthesis of connective tissue components, -prevent the fibrotic changes formation in tissues. To restore microcirculation and normalize connective tissue processes in the inflammation area, the Bovhyaluronidaze azoximer (lat.: Bovhyaluronidazum azoximerum) was included in the complex therapy of intervention groups animals. The Bovhyaluronidaze azoximer contains an immobilized hyaluronidase — hyaluronidase conjugate with a high-molecular carrier. Hyaluronidase hydrolyzes the hyaluronan (high-molecular hyaluronic acid) to tetrasaccharides [18], which reduces the viscosity of the connective tissue extracellular matrix, thereby increasing the permeability of tissues, facilitating the fluids movement in interstitial spaces and preventing pathological growth of connective tissue. In addition, the high-molecular carrier, being an activated derivative of poly-1,4-ethylene piperazine Noxide, possesses its own pharmacological activity and regulates the synthesis of inflammatory mediators interleukin-1 (IL 1) and tumor necrosis factor-alpha (TNFα), increases the humoral immune response and resistance to infections [19]. In our study, the proportion of animals in the intervention group with postoperative complications was 0.10. In the control group, the proportion of animals with postoperative complications was 0.55 [20]. Using the Pearson's Chi Squared Test (χ2 ) to compare the frequency of urethral and bladder surgery complications with Bovhyaluronidaze azoximer during postoperative period, it was found that there is a statistically significant relationship between the use of Bovhyaluronidaze azoximer and the frequency of postoperative complications, regardless of the type of surgery. Considering that a preoperative urinary tract infection is associated with postoperative complications development [21], it is reasonable to use Bovhyaluronidaze azoximer during the animal preoperative preparation in order to shorten the duration of the antimicrobial course and reduce the inflammatory process severity. Due to the anti-edematous and immunostimulating action of Bovhyaluronidaze azoximer as part of purulentinflammatory processes complex therapy, the median wound cleansing from purulonecrotic masses duration is reduced by 2.29 days. The median duration of edema reduction is reduced by 1.57 days and the median number of necessary wound management is reduced by 5.00 manipulations [22]. When comparing the results obtained in the intervention and control groups using the Mann-Whitney U Test, the p-value is significantly lower than the significance level adopted in our study (p=0.05), which indicates that the differences are statistically significant. Currently, we continue research in this direction and form an intervention group of animals in which systemic antibacterial therapy is not used as a part of purulentinflammatory processes treatment, and topical treatment is combined with the Bovhyaluronidaze azoximer in the form of rectal suppositories. Fig. 2 shows an abscess of the left perianal sinus in a 3-year-old male Chihuahua, admitted to the clinic with complaints of restlessness and severe pain in the perianal region. It was decided not to use a systemic antimicrobial in this animal. The course of Bovhyaluronidaze azoximer was reduced to 4 administrations, since by the 4th day of use the edema had completely subsided; the abscess cavity was completely cleansed. The abscess cavity was completely healed by the 7th day of treatment (Fig. 3). Fig.2 Abscess of the left perianal sinus (a lancing of abscess was performed) Because the hyaluronidase in Bovhyaluronidaze azoximer is covalently attached to a high-molecular carrier, the therapeutic doses are small enough to avoid allergic reactions. This, in turn, makes Bovhyaluronidaze azoximer the first-choice even if the patient is in a serious condition at the time of the operation, for example, with a urethral injury before surgery, extensive hematomas, infiltration of periurethral and perirectal tissues, 4 BIO Web of Conferences 30, 06007 (2021) https://doi.org/10.1051/bioconf/20213006007 ILS 2020
subcutaneous tissue, and intermuscular spaces of the pelvic muscles with urine [23]. Fig 3. The same animal as in Fig. 2, on the 7th day of treatment 4 Сonclusion This study showed that Bovhyaluronidaze azoximer has an anti-inflammatory effect, reduces the severity of edema and pain reactions and is able to prevent reactive growth of connective tissue as well as fibrosis development and complications of surgical intervention both in the short and long term. The use of the Bovhyaluronidaze azoximer during the pre- and postoperative period of operations on the urethra and bladder can quickly reduce edema and inflammation of the surgical wound tissues and tissues around the urethrostomy. It also prevents coarse scarring of the postoperative suture between the urethra mucous membrane and the skin, which leads to the urethrostoma closing and/or urethral strictures. As a result, the use of the Bovhyaluronidaze azoximer statistically significantly reduces the incidence of postoperative complications. The results of our studies indicate that Bovhyaluronidaze azoximer statistically significantly reduces duration of treatment, accelerates the wounds and cavities purification from purulent-necrotic contents, significantly reduces edema and tissue inflammation, helps restore microcirculation, and increases the bioavailability of antibacterial agents in the infection site. This allows reduce the duration of antibiotic courses (and in some cases to completely abandon antibiotic therapy) and prevent the formation of chronic infection site. Due to the covalent binding of hyaluronidase to a highmolecular carrier Bovhyaluronidaze azoximer has a prolonged action and resistance to denaturing agents which makes it convenient to use (which, in turn, increases the compliance of animal owners), and the likelihood of allergic reactions is reduced because of enzyme low therapeutic doses. In addition, as a result of hyaluronidase conjugation, it became possible to use the Bovhyaluronidaze azoximer in the acute phase of the inflammatory process when antibiotic therapy is conducted. Consequently, the bioavailability of antibacterial drugs increased and that also makes it possible to reduce the antimicrobial courses duration without loss of therapy effects. References 1. L. K.Vestby, vol. Gronseth, R. Simm, and L. L. Nesse, Antibiotics-Basel, 9, 29 (2020). 2. C. O'Meara, B. L. Boyanton, D. Spurlin, and C. F. Carpenter, Infectious Diseases in Clinical Practice, 24, 112–114 (2016). 3. V. D. Palumbo, P. Tralongo, B. Di Trapani, F. Carini, and G. Tomasello, Clinica Terapeutica, 170, E328–E331 (2019). 4. C. Nhan, A. Bezdjian, S. Saha, S. Prakash, L. H. P. Nguyen, and S. J. Daniel, Journal of Otolaryngology-Head & Neck Surgery, 46 (2017). 5. M. E. Ahmed, S. H Seddiq., R. Q. Basi, G. A. Abdulhasan, and A. Q. Al-Awadi, Research Journal of Pharmaceutical Biological and Chemical Sciences, 9, 1186–1196 (2018). 6. Z. Hadisi, J. Nourmohammadi, and S. M. Nassiri, Int. J. Biol. Macromol., 107, 2008–2019 (2018). 7. R. Dorsch, C. von Vopelius-Feldt, G. Wolf, R. S. Mueller, R. K. Straubinger, and K. Hartmann, Tieraerztliche Praxis Ausgabe Kleintiere Heimtiere, 44 , 227–236 (2016). 8. S. Teichmann-Knorrn and R. Dorsch, Tieraerztliche Praxis Ausgabe Kleintiere Heimtiere, 46, 247–257 (2018). 9. GOST R 58090-2018 “Clinical examination of unproductive animals. General requirements”, 2018. 10. Daniel D. Smeak, Proceedings of the North American veterinary conference, 1452–1454, 2006. 11. Clara S.S. Goh and Howard B. Seim, Today’s Veterinary Practice, 43–49 (2014). 12. Karen M. Tobias, , NAVC clinician’s brief, 19– 22 (2007). 13. B. S. Semenov, and A. V. Nazarova, “Perineal urethrostomy male cats: Pros and Cons”, International bulletin of Veterinary Medicine, 2, 130–135 (2018). 14. M. T. Watson, R. Y. Roca, A. H. Breiteneicher, and R. H. Kalis, Journal of Feline Medicine and Surgery, 22, 399–403 (2020). 15. R. P. Sousa, D. C. S. Nunes-Pinheiro, K. O. Sampaio, E. C. B. da Silva, G. Cavalcanti, and M. da Cunha, Journal of Feline Medicine and Surgery, in press. 16. Z. Nikousefat, M. Hashemnia, M. Javdani, and A. Ghashghaii, Veterinary Research Forum, 9, 199–203 (2018). 17. A. V. Zaitsev, Effective Pharmacotherapy, 2, 50–56 (2018). 18. R. U. Habriyev, N. O. Kamayev, T. I. Danilova, and E. G. Kakhoyan, Biomedical Chemistry, 62, 82–88 (2016). 5 BIO Web of Conferences 30, 06007 (2021) https://doi.org/10.1051/bioconf/20213006007 ILS 2020
19. Yu. G. Alyayev, Ye. V. Lartsova, and L. G. Spivak, Effective Pharmacotherapy, 49, 4–8, 2015. 20. A. A. Stekolnikov, A. V. Nazarova, B. S. Semenov, and T. Sh. Kuznetsova. International bulletin of Veterinary Medicine, 4, 158–165, 2019. 21. D. L. Frem, H. A. Hottinger, S. L. Hunter, and N. J. Trout, Javma-Journal of the American Veterinary Medical Association, 251, 935–940 (2017). 22. A. V. Nazarova, B. S. Semenov, A. A. Stekolnikov, and T. Sh. Kuznetsova, “The use of Bovhyaluronidaze azoximer preparation in the treatment of suppurative wounds”, BIO Web of Conferences, 23. A. V. Nazarova, B. S. Semenov, A. V. Bokarev, and A. Yu. Nechaev, Collection of scientific papers of the Ninth international interuniversity conference on clinical veterinary medicine, 97– 110 (2019). 6 BIO Web of Conferences 30, 06007 (2021) https://doi.org/10.1051/bioconf/20213006007 ILS 2020