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structure in normal and in terms of pathology - acute pancreatitis.
2. The histological structure of pancreatic drugs indicates cyto- and morphometric changes in the exocrine and endocrine parts. The results of morphometric studies of the exocrine part indicate a tendency to increase (p <0.001) in 1.12 times the exocrine parenchyma of cats, equal to 48257.09 ± 135.06 ^m2 or 97.89 ± 0.04%. The endocrine part of the gland, in contrast, was reduced by 39.56 ^m2 times and amounted to 6771.88 ± 274.06 ^m2. Such morphological parameters in control animals were 42989.03 ± 141.05 ^m2 -96.11 ± 0.05%% and 6811.44 ± 312.04 ^m2 - 3.89 ± 0.03%.
References
1. Andreeva, S.D., & Kirillovyh A.S. (2013). Ul'tramikroskopicheskoe stroenie mikrocirkuljator-nogo rusla podzheludochnoj zhelezy pri ostrom destruktivnom pankreatite. Vestnik veterinarii. Materi-aly Mezhdunarodnoj nauchno-prakticheskoj internet-konferencii, posvjashhènnoj 65 letiju Stavropol'skogo GAU, «Sovremennye tendencii v veterinarnoj medicine». Stavropol': SGAU, 16, 30-33.
2. Horalskyi, L.P., Khomych, V.T., & Kononskyi, O.I. (2019). Osnovy histolohichnoi tekhniky i mor-fofunktsionalni metody doslidzhennia u normi ta pry patolohii: navch. posib. Zhytomyr: Polissia, 288.
3. Didenko, I.S. (2017). Zminy histolohichnoi struktury pidshlunkovoi zalozy za umov aloksanovoi hiperhlikemii u shchuriv u vikovomu aspekti. Ukrain-skyi zhurnal medytsyny, biolohii ta sportu, 6(8), 17-20.
4. Dubych, I.M. (2008). Histomorfolohiia ta orha-nometrychni pokaznyky pidshlunkovoi zalozy tsutse-niat. Nauk. visnyk Lvivskoho natsionalnoho univer-sytetu veterynarnoi medytsyny ta biotekhnolohii im. S.Z. Hzhytskoho. Lviv, 10(3(38)), 73-76.
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5. Zinenko, D.Yu., & Berehovenko, I.M. (2008). Mikrotsyrkuliatorni y patomorfolohichni zminy u rozvytku eksperymentalnoho hostroho pankreatytu u shchuriv. Morfolohiia, 2(1), 33-40.
6. Tymoshenko, O.P. (2007). Informatyvnist son-ohrafichnoho doslidzhennia u diahnostytsi zakhvori-uvan pidshlunkovoi zalozy sobak. Visnyk Bi-lotserkivskoho derzh. ahrarnoho un-tu: Zb. nauk. prats. Bila Tserkva, 44, 101-103.
7. Katten, I. (2013). Sochetaniepankreatita s hol-angiogepatitom i vospalitel'nym zabolevaniem kishechnika (triadit) u koshek. Fokus, 23(2), 4-10.
8. Kravchenko, S.O., & Bobrova, V.V. (2018). Ul-trasonohrafichni zminy za hostrykh ta khronichnykh zapalen pidshlunkovoi zalozy u sviiskykh kotiv. Visnyk Poltavskoi derzhavnoi ahrarnoi akademii, 1, 138-142.
9. Ksenulis, P., & Shtainer, Y. (2009). Pankreatyt u koshek. Fokus, 19(2), 11-20.
10. Pankreatyt sobak. Monohrafiia: L.P. Ho-ralskyi, O.P. Tymoshenko, B.V. Borysevych ta in; Za redaktsiieiu L. P. Horalskoho. Zhytomyr: «Polissia», 215.
11. Gushhin, Ja.I. i dr. (2018). Sravnitel'naja mor-fologija podzheludochnoj zhelezy jeksperimental'nyh zhivotnyh i cheloveka. Laboratornye zhivotnye dlja nauchnyh issledovanij. Nauchno-prakticheskij zhurnal. Nauchno-prakticheskij zhurnal, 3, 33-48. doi: 10.29296/2618723X-2018-03-04
12. Tymoshenko, O.P., & Busel, Yu.M. (2009). Efektyvnist kompleksnoi diahnostyky pankreatytu v sobak, pidtverdzhena morfolohichnymy doslidzhen-niamy. Visnyk Poltavskoi derzh. ahrar. akad., 1, 87-93.
13. Akol, K.G., Washabau, R.J., Saunders, H.M., & Hendrick, M.J. (1993). Acute pancreatitis in cats with hepatic lipidosis. Journal of Veterinary internal Medicine, 7, 205-209.
Leskiv Kh Ya., Gufriy D.F., Gutyj B.V.,
Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies,
Pekarska, Str., 50, Lviv, 79010, Ukraine
Khalak V. I.
State Institution Institute of grain crops of NAAS, V. Vernadsky Str., 14, Dnipro, 49027, Ukraine
Hariv I.I., Martyshuk T. V., Guta Z.A.
Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies,
Pekarska, Str., 50, Lviv, 79010, Ukraine DOI: 10.24412/2520-6990-2021-794-12-15 THE EFFECT OF METHIPHENE, METHIONINE, AND PHENARONE ON THE HUMORAL PART OF THE IMMUNE SYSTEM OF PIGLETS IN EXPERIMENTAL CHRONIC NITRATE-NITRITE
TOXICOSIS
Abstract.
The article describes the dynamics of indicators of the state of humoral immunity ofpiglets in experimental nitrate-nitrite toxicosis. The subjects were 20 large white piglets three months old. The research was conducted at the Komarnivsky National Research Center of the Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies. According to the method of analog groups, one control and three experimental were formed. Feeding sodium nitrate to piglets at a dose of 0.3 g NO3'/kg. b.w. contributes to the development of chronic nitritenitrate toxicosis. Nitrates in a toxic dose for 90 days reduce the protective properties of the body. Under nitrate load, feeding pigs phenarone, methyphene, and methionine had a positive effect on piglets' humoral immunity, manifested by the high antimicrobial activity of serum.
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Keywords: Methyphene, phenarone, methionine, humoral immunity, nitrate-nitrite toxicosis.
Introduction. Many scientific works by domestic and foreign researchers are devoted to studying issues related to nitrate-nitrite toxicosis in animals and poultry. Among them in our country, the most fundamental researches are carried out by S.V. Bazhenov, Z.P. Skorodynsky, G.O. Khmelnytsky, A.Y. Mazurkevich, O.O. Malinin, D.F. Gufriy, O.I. Kanyuka, V.M. Hunchak and others. These scientists have identified the main links in the pathogenesis of nitrate-nitrite toxicosis with a different course in animals and birds, significantly identified features of metabolic processes and disorders of physiological functions of the body [1]. However, the mechanism of toxic effects of nitrates and nitrites on pigs has not been studied enough.
Still, an urgent problem in animals' chronic toxicosis is the development of veterinary drugs that can increase antioxidant activity and immune resistance of animals, normalize metabolic processes in tissues, restore the structure and function of organs and systems. Among them, various specific and nonspecific biologically active drugs have become widespread. One of them is phenarone, which delays lipid oxidation and reduces free radicals' content to preserve biologically active vitamin preparations and feed supplements. Phenarone is a complex compound containing 70% phenazanoic acid and 30% zeolite. In animals, phenarone stimulates the conversion of peroxides to inactive metabolites, which increases the nonspecific resistance of animals [6-8].
The mechanism of action of phenarone in chronic nitrate-nitrite toxicosis is associated with the direct effect of its components on the inhibition of the absorption of metabolites that have a toxic effect on cell membranes caused by nitrites. As a result of the adsorbing action of zeolite in the digestive tract, there is a decrease in the concentration of substances formed by nitrates and nitrites, which can be toxic to the body and enhance the processes of lipid peroxidation. This cause occurs by osmosis and diffusion of these substances through the capillaries of the small intestine's microvilli and their subsequent fixation on the sorbent granules [5]. By preventing the entry of toxic substances from the digestive tract into the blood and developing inflammatory reactions in the intestine, enterosorption indirectly contributes to the functional unloading of the liver, thus ensuring more efficient functioning of the antioxidant defense system. Phenosan acid is a direct-acting antioxidant that interacts directly with lipid peroxidation products and free radicals.
However, many authors [2] found that when nitrates enter animals' bodies in large quantities, phenarone did not have a proper corrective effect on the antioxidant defense system and neutralization of lipid peroxidation products. That prompted the development of a new drug that could perform a proper corrective function.
At the Department of Pharmacology and Toxicology of Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies Lviv, the antioxi-dant Methyphene was developed, which contains the drug phenarone and the amino acid methionine [3].
The methionine combines enzymatic and non-enzymatic systems of antiradical protection of biological cell membranes. It converts neutral fats into phospho-lipids, which stabilize subcellular membranes, thus providing antioxidant protection and increasing hepato-cytes' resistance against the toxic effects of harmful substances, including nitrites. Methionine also maintains calcium homeostasis and prevents the development of the fatty liver disease.
Therefore, it is known from the literature that nitrate-nitrite toxicosis in animals uses drugs that have antioxidant properties because nitrate-nitrite toxicosis is the depletion of the antioxidant system and the growth of lipid peroxidation products. The proposed methods of treatment of animals in this intoxication apply only to poultry and cattle. Still, this toxicosis also affects pigs, so the urgent problem is to study the path-ogenesis of nitrate-nitrite toxicosis in swine and develop an effective method of treatment and prevention of nitrate-nitrite intoxication [4]. The use of known remedies is insufficiently studied in pigs. That is why the research of phenarone, methiphene, and methionine in nitrate-nitrite toxicosis in swine is relevant.
The aim of the study. To investigate the effect of phenarone, methiphen, and methionine on the humoral part of the immune system of pigs in experimental nitrate-nitrite toxicosis.
Materials and methods of research. The subjects were 20 large white piglets three months old. The study was conducted at the Komarnivsky National Research Center of the Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnologies. According to the method of analog groups, one control and three experimental were formed. The scheme of the experiment is given in table 1.
Table 1
Groups of animals The dose of the drug
C Piglets were fed sodium nitrate at a dose of 0.3 gNO3_/kg body weight once a day during the studies
E1 Piglets were fed methionine at a dose of 4 mg/kg + sodium nitrate at a dose of 0.3 gNO3_/kg body weight once a day during the studies
E2 Piglets were fed phenarone at a dose of 1.20 mg/kg + sodium nitrate at a dose of 0.3 gNO3_/kg body weight once a day during the studies
E3 Piglets were fed methiphen at a dose of 0.9 mg/kg + sodium nitrate at a dose of 0.3 gNO37kg body weight once a day during the studies.
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Blood for investigation was taken from the cranial vena cava at the beginning of the experiment and 10, 30, 60, 90 days. From humoral indicators of resistance bactericidal activity of blood serum (BABS) was investigated by the method of O.V. Smirnova, T.A. Kuzmina (1966), lysozyme activity of blood serum (LABS) - photo electro colorimetric method, and titer of normal antibodies of blood serum (TNABS) in agglutination reaction (Chumachenko V.E., etc., 1990).
Research results.
The investigation of the effects of phenarone, me-thiphen, and methionine on humoral immunity in nitrate-nitrite toxicosis in pigs is shown in table 2.
It is known that specific macromolecules provide humoral immunity that functions in the internal fluids of animals. Blood plasma contains particular proteins that can neutralize microorganisms and toxic products of their activities that enter body fluids. The function of the immune system is to recognize genetically foreign
antigens and specifically respond to them. Its primary purpose is to neutralize and destroy those antigens that stimulate the immune response.
As shown in Table 2, in chronic nitrate-nitrite toxicosis in piglets, serum antimicrobial activity in the first days of the study increased, as indicated by grew up of the bactericidal and lysozyme activity of serum in sick animals. Thus, on the 10th day, BABS increased by 11%, while the LABS in this period grew up by 4%. Subsequently, the antimicrobial activity of the serum of piglets fed sodium nitrate in a toxic dose began to decline gradually, indicating the suppression of the physiological state of the humoral immune system. On the 30th day, the C group's BABS reached 25.13+0.65%, and the LABS - 39.41+0.60%. The lowest antimicrobial activity was found on the 60th day, compared with the initial values, BABS decreased by 9% and LABS -by 6.8%.
Table 2
The indicators of humoral immunity of piglets feeding methionine, phenarone, and methyphene under conditions of chronic nitrate-nitrite toxicosis (M±m, n=5)
Indicators Groups of animals
Start day 10th day 30th day 60th day 90th day
Bactericidal activity (BABS) % C 26.44+0.49 29.41+0.60 25.13+0.65 24.04+0.54 24.83+0.50
E1 25.79+0.49 28.79+0.50 25.20+0.49 24.91+0.50* 25.05+0.56
E2 25.91+0.48 28.64+0.65 25.26+0.50 25.53+0.52** 25.42+0.49
E3 26.28+0.50 28.55+0.63 26.06+0.61 26.41+0.55** 26.35+0.61
Lysozyme Activity (LABS) % C 41.12+0.61 42.89+0.63 39.41+0.60 38.31+0.72 40.21+0.70
E1 41.33+0.70 41.64+0.60 39.86+0.65 39.92+0.54*** 39.99+0.70
E2 41.24+0.61 41.60+0.75 40.15+0.63 40.22+0.60*** 40.76+0.65
E3 41.08+0.60 41.55+0.64 41.28+0.60 41.12+0.75*** 41.20+0.60
CIC, mM / ml C 79.28+3.13 79.85+3.10 80.91+3.11 84.32+3.13 80.86+3.11
E1 79.31+3.10 79.34+3.13 79.74+3.13 82.86+3.10 79.91+3.10
E2 79.39+3.10 79.41+3.11 79.68+3.12 80.75+3.13** 79.42+3.10
E3 79.15+3.11 79.19+3.10 79.42+3.12 79.27+3.11 79.20+3.12
The degree of probability, compared with the control group: * -p < - 0,05; ** -p < - 0.02; *** -p < 0.001
On the 90th day of the research in the C group was found an increase in BABS and LABS, as indicated by the adaptation of piglets to long-term sodium nitrate intake, but comparing the values with blood taken at the beginning of the experiment, antimicrobial activity of serum was lower.
Under physiological conditions, the formation and presence of circulating immune complexes (CIC) in fluids manifest the body's immune response to antigen and an essential factor in ensuring immunity. The formed immune complexes under these conditions circulate in lymph and blood for some time; then, there is their detection. Simultaneously, the CIC can trigger chains of pathological changes because their prolonged circulation, even with a slight increase in body fluids, leads to accumulation in tissues. This, in turn, causes increased aggregation and adhesion of platelets, which leads to disruption of microcirculation and obliteration of blood vessels of the hemomicrocirculatory tract, tissue damage and necrosis. Formed CIC interacts with almost all blood cells, with complement, and receptors of many cells of organs and tissues. The interaction of
the CIC with immunocompetent cells leads to the modulation of the immune response.
In studying the values of humoral immunity in sick piglets in the blood, the level of circulating immune complexes (CIC) on the 30th day of the experiment is 80.91+3.11 mM/ml against 79.28+3.13 mm/ml in clinically healthy. On the 60th day, the highest CIC level was established, where, according to the initial values, it increased by 6%. High levels of CIC in the serum indicate the suppression of the body's immune system due to the attachment of specific antibodies to the products of metabolism in chronic nitrate-nitrite toxicosis.
Therefore, the simple detection of high CIC levels without clinical signs, anamnesis data indicates only the stimulation of piglets' immune response, which is aimed at removing from the body genetically foreign antigens.
In studying the values of humoral immunity of piglets fed methionine, phenarone, and methyphene for three months, high antimicrobial activity of blood serum was found.
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The bactericidal activity of serum of piglets fed methionine and sodium nitrate was 28.79+0.50% on the 10th day of the experiment and 25.20+0.49% on the 30th day, respectively. On the 60th day, BABS in this experimental group decreased slightly but, compared with the C group, risen by 4%. On the 90th day, this figure reached the limits of the physiological norm.
Feeding phenarone and methiphene contributed to a better normalization of serum bactericidal activity than the use of methionine. Thus, on the tenth day of the experiment, we note an increase in the studied indicator, respectively, by 10 and 9% compared to the initial values. Still, relative to the C group of animals, this figure was slightly lower. On the 30th day, serum bactericidal activity in both experimental groups was in the range of 25.26+0.50 - 26.06+0.61%. On the 60th day, the BABS was the highest in the experimental group of animals fed methiphen. According to the C group, it raised by 10%. On the 60th day, BABS reached the initial values' limits.
Lysozyme activity of blood serum of piglets on the 10th day in all experimental groups was slightly lower than the control group of animals. The E1 group was 41.64+0.60%, in the E2 group - 4, 60+0.75%, in the E3 group - 41.55+0.64%, while in the C group it was 42.89+0.63%.
In subsequent periods of the study, LASC in the experimental groups of piglets continued to decline, but compared with the control group of animals, it increased slightly, respectively, in piglets fed methionine - by 1.1%, in piglets fed phenarone - by 2%, in piglets fed methyphene - by 5%. On the 60th day, the most probable changes in the above indicator of the experimental groups of animals compared with the control group were found. Thus, in the E1 group, LABS grew by 4%, in the E2 group - by 5%, and in the E3 group, respectively, by 7%. On the 90th day, LASK continued to grow, but it reached the initial values only in the experimental group of animals treated with methiphen, where, respectively, it was 41.20+0.60%.
In general, the results indicate a specific stimulating effect of methionine, phenarone, and methiphene on humoral immunity activity in blood, namely BABS and LABS, when added to compound feed to piglets that were nitrated for three months
Having established that the CIC increases significantly in chronic nitrate-nitrite toxicosis, we tested an-tioxidant drugs with hepatoprotective and detoxifying effects. In piglets treated with these remedies before nitrate and nitrite poisoning, the CIC content did not change compared to intact animals. The absence of changes in CIC content in the body of poisoned piglets
treated with phenarone and methiphene may indicate the intensity of detoxification processes and thus reduce antigens' formation, which leads to stabilization of the content of CIC in the blood.
Conclusions
1. Feeding sodium nitrate to piglets at a dose of 0.3 g NO3~/kg. b.w. contributes to the development of chronic nitrite-nitrate toxicosis. Nitrates reduce the body's protective properties in a toxic dose; when they enter the animal's body for 90 days
2. Under nitrate load, feeding pigs phenarone, methyphene, and methionine had a positive effect on piglets' humoral immunity, manifested by the high antimicrobial activity of serum.
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