State Scientific-Research Control Institute of veterinary medical products and feed additives

1. 7th Framework Programme , the project ACOUSTICS (ITEA- 0941 ): «Acoustic wave application for the analysis of the quality and safety of porous food and non-food matrices» – «ACOUSTICS» program ITEA, which were developed and implemented innovative methods and techniques detect mycotoxin DON in feeds and feed raw materials. In the final phase of work – conducted the validation acoustic device, developed in cooperation with Lithuanian partners through comparative tests with traditional methods of determination of mycotoxins.

2. Immunological research the effect of probiotics (Woogene B & G Co, Ltd, SEOUL OFFICE: R. NO. 1504 , Ace Hitech City, 1 -Dong, # 55-20, Munrae-dong 3 -Ga, Yeongdeungpo-gu, Seoul, Korea 150-972 ) on animals, 2012.

Studies conducted on the private farm KT “Ego” Zhovkva district, Lviv region, 1,200 heads of broiler chickens cross “Cobb-500,” the daily age, divided into four groups of 300 animals each. To determine the probiotic efficacy «PROBION» and the study of its effects on the body of chickens used product with feed in different concentrations: 1 group received probiotic «PROBION» in a dose of 1 g / kg, Group 2 – «PROBION» in a dose of 0.5 g / kg, group 3 chickens received probiotic-analog “Bio plus 2B” at a dose of 0.4 g / kg, group 4 served as control. Feed fed according to standards recommended by the company for the cross “Kobb-500.”

Vaccination of chickens conducted : against infectious bronchitis of hens – on 12th days, against Gumboro disease ( BG ) – on 18th and 25th days, and against Newcastle disease (BN ) – on 21 day of the experiment. Dynamics of changes in body weight of broilers was studied by individual weighing under the scheme: 8 , 15, 22, 28, 36 and 43 days.

Bird staying on the floor with free access to feed and water. On the 15th and 30th days of each group were collected 50 goals, 43 days of each group were selected 200 goals.

Clinical trials of the drug Probion-forte conducted on pigs in NNVTS “Komarnivskyy” Gorodok district, Lviv region. Of the 120 heads of pigs breed ‘Large White’, 28 days old, was formed four groups of 30 goals each. To determine the efficiency, safety probiotic Probion-forte production Woogen, Korea and the study of its effects on the body piglets fed with food preparation in different concentrations: one group received probiotic Probion-forte dose of 1 g / kg of feed (10 weeks); Group 2 – Probion-forte dose of 1 g / kg (6 weeks) and 0.5 g / kg of feed (4 weeks); Group 3 pigs receiving probiotic analog Bio plus 2B, manufactured by Biochem, Germany, which was used at a dose of 0.4 g / kg of feed for 10 weeks; Group 4 served as control. Feed fed according to standards recommended for breeds “Great White” with age. Veterinary treatment carried out according to the developed economy in the scheme. Dynamics of changes in body weight of pigs was studied by weighing individual under the scheme: 1, 14, 28, 42, 56 and 70 days of experiment and at the end of the feeding period. Performance measures at slaughter animals. The animals were kept in cages for 15 goals each, with free access to food and water. 42 day experiment (6 weeks) 1, 3 and 4 groups and 70 day experiment (10 weeks) 1, 2, 3 and 4 selected in each of 10 goals for hematological, pathological and microbiological studies. Diagnostic slaughter was carried out on 42 and 70 day experiment, 3 heads of each group.

For pathological and microbiological studies. With each animal after slaughter samples were collected intestinal wall, they were fixed in 10% neutral formalin, Buyena holder, followed by pouring paraffin. Paraffin sections were stained with hematoxylin -eozynom by the conventional method. In the finished histological preparations were determined: villi length, crypt depth and their relationships, cellular composition and size tsekalnoyi tonzyly. Microscopy was performed using a microscope OLIMPUS SH-41 and morphometric program DP-SOFT.

Factors mass of internal organs was defined as the ratio of the mass in grams of the organ to body weight in kilograms.

The selected material for histological studies were fixed in 10% neutral formalin solution -th and tissue dehydration – in ascending series of alcohol (70, 80, 90, 96 and 96-II), followed by sealing in chloroform and hloroformparafini and fill in paraffin. After pouring the material prepared in paraffin sections 4-5 microns thick on Sannomiya microtome (ICP-2) and stained with hematoxylin-eosin.

For microbiology samples were taken vmistymoho blind guts. In vmistymoho intestinal samples studied species quantitative and qualitative composition of microflora by dilution and seeding microorganisms on selective medium. The identification of microorganisms was carried out by conventional methods. Statistical analysis of the results was carried out on a computer using Microsoft Excel.

Material for hematological and biochemical studies served as a selected sample of blood. Blood in broiler chickens were taken from the subclavian vein in pigs with cranial vena cava. In heparynizovaniy measured blood concentrations of hemoglobin, hematocrit, number of erythrocytes, leukocytes, leykoformulu, phagocytic activity of neutrophils (FAN) and phagocytic index (FI). Serum (SC) – the concentration of total protein and its fractional composition, lizotsymnu (LASK) and bactericidal (BASK) activity serum and circulating immune complexes (CIC), the activity alaniaminotransferazy (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP ), the content of total cholesterol, total lipids, triacylglycerols.

Determination of morphological, biochemical blood parameters in accordance with generally accepted methods using standard certified test kits and automated hematology analyzer 3- DIFF Mythic TM 18 and biochemical analyzer HumaLyzer-3000.

These hematological, biochemical and pathological studies statistically treated calculation of arithmetic variables (M), mean square error (m) and the probability of a difference (R) between indicators. Digital values expressed in units of SI system. Statistical analysis of the results of research conducted by the method described IA Oyvinym (1960), using the program “Excel-97” for Windows (TF Lapkin, 1990). Probability differences between indicators assessed by Student’s test (P <0.05).

Similar studies conducted in young cattle.

3. Nifursol marker metabolite (DNSH) determination by LC-MS/MS: development of the method and testing on incurred chicken tissues samples

Goal: to develop and validate new simple and rapid LC-MS/MS method for the determination of total residues of nifursol marker metabolite (3,5-dinitrosalicylic acid hydrazide, DNSH) in broiler chicken tissues after the derivatization with 2-nitrobenzaldehyde; to carry out in vivo experiment in order to obtain naturally incurred samples of chicken tissues and parenchymal organs; to test the performance of the new method on incurred samples.

Nifursol belongs to the class of nitrofurans – synthetic broad-spectrum antibacterial drugs with five-membered ring heterocycle in their structure. Nifursol was the last authorised substance for the prevention of black head disease of poultry and has been prohibited for use as a feed additive by Council Regulation 1756/2002/EC. Long-term study on experimental animals revealed carcinogenic and mutagenic properties of nitrofurans metabolites, which was the reason why these drugs were banned for the treatment of food-producing animals within the EU and many other countries. Nifursol, like other nitrofurans, rapidly metabolises in vivo and forms highly stable major protein-bound metabolite 3,5-dinitrosalicylic acid hydrazide (DNSH), which is used as the marker residue of parent drug illegal use. According to the Commission Regulation (EU) 2019/1871, the EU reference point for action (RPA) for DNSH in food products of animal origin is 0.5 μg kg-1. Critical issue of confirmatory determination of nitrofuran metabolites bound residues in animal tissues is complicated and time consuming preliminary washing step by organic solvents to remove free metabolites and part of matrix components. So the elaboration of an effective method for DNSH total residues assay in animal tissues and its approval on incurred samples is a question of current interest.

The current project is performed in the collaboration with R-Biopharm Nederland B.V.