Kazakh National Agrarian University UDC 619:636.087.7 on the Rights Of

Kazakh National agrarian university

UDC 619:636.087.7 On the rights of manuscript

ABDIGALIYEVA TOLKYN BAKYTOVNA

Veterinary sanitary assessment of poultry products while using feed additives based on vermiculite

6D120200 – Veterinary sanitation

A dissertation submitted for the degree of Doctor of Philosophy (Ph.D)

The domestic scientific advisers Doctor of veterinary sciences, professor Sarsembayeva N.B., Candidate of veterinary sciences, assistant professor Ussenbayev A.E.

Foreign scientific adviser Doctor of chemical sciences, professor of Institute Plant protection (Poland) Lozowicka B.

The Republic of Kazakhstan Almaty, 2018

CONTENTS

NORMATIVE LINKS 4 DEFINITIONS 6 DESIGNATIONS AND ABBREVIATIONS 8 INTRODUCTION 10 1 REVIEW OF LITERATURE 12 1.1 Development of poultry farming in Kazakhstan 12 1.2 Veterinary and sanitary examination of poultry products 16 1.3 Food and biological value of meat and eggs 21 1.4 Mineral feed additives and using them in poultry farming 25 1.5 Vermiculite-its composition and influence to the organism of animals 27 and birds 2 OWN RESEARCHES 31 2.1 Materials and methods 31 3 RESEARCH RESULTS 47 3.1Veterinary sanitary requirements and technological properties of 47 vermiculite from Kulantau deposit for feed preparation 3.2 Research the mineral components and microstructure of experimental 55 vermiculite 3.3 Veterinary and toxicological evaluation of vermiculite 58 3.4 The effect of vermiculite on the quality of fishmeal 60 3.5 Study the content of residual amounts of pesticides in feed and feed 68 additives based on vermiculite 3.6 Hematological and biochemical parameters of the blood of birds when 77 using feed additives based on vermiculite 3.7 Veterinary and sanitary assessment of the quality of meat and eggs when 80 used the feed additives based on vermiculite in the composition 3.7.1 Effect of feed additives based on vermiculite on the growth and 80 productivity of broiler chickens 3.7.2 The qualitative characteristics of eggs when used feed additives based on vermiculite 82 3.7.3 Organoleptical and quality parameters of meat and eggs when used 83 feed additives based on vermiculite 3.7.4 Nutritional value and chemical composition of the meat and eggs of 91 experimental birds 3.7.5 Analysis of the fatty acid composition of meat and eggs when used 95 feed additives based on vermiculite 3.7.6 Aminoacid composition of meat and eggs 104 3.7.7 Vitamin content of the meat and eggs while used feed additives containing vermiculite 109 3.7.8 Mineral content of meat and eggs 112 3.8 Morphological and histological changes of broiler chickens` meat while 115 using vermiculite in feed 2

4 GENERALIZATION AND EVALUATION OF RESEARCH RESULTS 117 CONCLUSION 122 PRACTICAL PROPOSALS 124 REFERENCES 125 APPENDIXES 140

NORMATIVE REFERENCES

In this dissertation references are used to the following standards:

GOST 31962-2013 Chicken meat (carcasses of chickens, broiler-chickens and their parts). Specifications GOST 18292-85 Slaughter poultry. Specifications GOST 7702.0-74 Poultry meat. Methods of sampling. Organoleptic methods of quality assessment GOST 32008-2012 Meat and meat products. Determination of nitrogen content (reference method) GOST 9793-74 Meat products. Methods for determination of moisture content GOST 23042-86 Meat and meat products. Methods of fat determination GOST 31654-2012 Food chicken eggs. Specifications GOST 32149-2013 Food products of commercial poultry eggs processing. Microbiological analysis methods GOST 19496-93 Meat.Method of histological investigation GOST 51479-99 Meat and meat products. Method for determination of moisture content GOST 23392-78 Meat. Methods for chemical and microscopic analysis of freshness GOST 53642-2009 Meat and meat products. Determination of total ash GOST 25011-81 Meat and meat products. Methods of protein determination GOST R55483-2013 Meat and meat products. Determination of fatty acids composition by gas chromatography GOST R 55482-2013 Meat and meat products. Method for determination of water-soluble vitamins GOST 2116-2000 Meal from fish, marine mammals, crustaceous and invertebrates. Specifications GOST 13496.18-85 Mixed fodder and fodder raw stuff. Methods for determination of fat acid value GOST 13496.12-98 Mixed fodder, raw mixed fodder. Method for determination of total oxidity GOST 13496.3-92 Compound feeds, raw material. Methods for determination of moisture GOST 13496.4-93 Fodder, mixed fodder and animal feed raw stuff. Methods of nitrogen and crude protein determination ISO 5983-1:2005 Feeds, mixed feeds and raw material. Determination of mass fraction of nitrogen and calculation of mass fraction of crude protein. Part 1. Kjeldahl method GOST 25336 – 82 Laboratory glassware and equipment. Basic parameters and dimensions

GOST 13502-86 Paper packets for loose products. Specifications ISO 24333-2009 Cereals and cereal products. Sampling GOST 13496.20-87 Mixed fodders, raw stuff for mixed fodders. Method for determining residual quantities of pesticides ISO/IEC 17025:2005 Requirements for testing laboratories (centers) of railway products CAC/MRL 1 Maximum permissible level of pesticides. GOST 26928-86 Food-stuffs. Method for determination of iron

DEFINITIONS

This dissertation uses the following terms with corresponding definitions: Aluminosilicates - are minerals composed of aluminium, silicon, and oxygen, plus countercations. They are a major component of kaolin and other clay minerals. Amino acids - are biologically important organic compounds composed of amine (-NH2) and carboxylic acid (-COOH) functional groups, along with a side- chain specific to each amino acid. An eggshell - is the outer covering of a hard-shelled egg and of some forms of eggs with soft outer coats. Animal feed - is food given to domestic animals in the course of animal husbandry. A Breed - a kind of the pets and birds (or plants) differing in some signs from animals and birds (or plants) the same look, family. A fatty acid - is a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. A vitamin - is an organic compound and an essential nutrient that an organism requires in limited amounts. Biological value - is a measure of the proportion of absorbed protein from a food which becomes incorporated into the proteins of the organism. Chemical composition - is a conventional designation of the chemical composition and structure of compounds with the help of symbols of chemical elements, numerical and auxiliary signs. Chromatography - is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. The various constituents of the mixture travel at different speeds, causing them to separate. The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation. Diet - daily forage, the giving made of various forages taking into account need of animals for nutrients. Egg white - is the name for the clear liquid (also called the albumen) contained within an egg. Fat – is triglycerides, are esters of three fatty acid chains and the alcohol glycerol. Feed additives - are products used in animal nutrition for purposes of improving the quality of feed and the quality of food from animal origin, or to improve the animals’ performance and health, e.g. providing enhanced digestibility of the feed materials. Fight - eggs, with the damaged shell without leak. Ion exchange - is an exchange of ions between two electrolytes or between an electrolyte solution and a complex. In most cases the term is used to denote the

6 processes of purification, separation, and decontamination of aqueous and other ion- containing solutions with solid polymeric or mineral ion exchangers. Minerals - are solid natural formations that form part of the rocks of the Earth, the Moon and some other planets, as well as meteorites and asteroids. Nutritional value - is a complex of properties of food products that provide physiological needs of a person in energy and in basic nutrients. Organoleptic properties - are the aspects of food, water or other substances that an individual experiences via the senses - including taste, sight, smell Ovoscope - equipment for determining the quality of eggs by their translucence. Pesticides - are substances that are meant to control pests or weeds. The term pesticide includes all of the following: herbicide, insecticide, insect growth regulator, nematicide, termiticide, molluscicide, piscicide, avicide, rodenticide, predacide, bactericide, insect repellent, animal repellent, antimicrobial, fungicide, disinfectant (antimicrobial), and sanitizer. Proteins - are large biomolecules, or macromolecules, consisting of one or more long chains of amino acidresidues. Sorbents - are insoluble materials or mixtures of materials used to recover liquids through the mechanism of absorption, or adsorption, or both. Absorbents are materials that pick up and retain liquid causing the material to swell (50 percent or more). Adsorbents are insoluble materials that are coated by a liquid on its surface. To be useful in combating oil spills, sorbents need to be both oleophilic (oil-attracting) and hydrophobic (water-repellent). The table eggs - eggs, which period of storage at a temperature from 0 °C to 20 °C - no more than 25 days, and eggs which were stored at a temperature from minus 2 °C up to 0 °C - no more than 90 days. The cooled eggs - the eggs cooled in rooms where temperature is artificially maintained from minus 2 °C up to 5 °C. The mass of egg - is the major physical indicator of nutrition and commodity value defining efficiency of a bird. Veterinary and sanitary examination - is a branch of the veterinary industries, which studies the methods for sanitary and hygienic research of food and technical raw materials of animal origin and defines the rules for their veterinary and sanitary evaluation. Washed eggs - eggs, processed with special detergents, approved for use in the territory of the state that adopted the standard.

DESIGNATIONS AND ABBREVIATIONS

AIC - agro-industrial complex cm - centimeter DDT - 4,4 '-dichlorodiphenyl-trichloroethane EU - European Union FAO -Food and Agriculture Organization FID - flame-ionization detector FM - fishmeal g - gramm g/l - gram per litre GC - a gas chromatograph GOST - government standard h – hour HCH - hexachlorocyclohexane KazNAU – Kazakh National agrarian university kg - kilogram KJIC - Kazakhstan-Japan Innovation Center LD - lethal dose LIA - lioneic acid LLP – A limited liability partnership is a partnership in which some LOD - limit of detection LOQ - limit of quantification MA - Ministry of Agriculture mg - milligram min - minute mkg - mikrogram ml- milliliter mln. - million mm - millimeter MPC - maximum permissible concentration MRL-maximum permissible level MUFA - monounsaturated fatty acids MYA - myristic acid n - normal ND - normative documents NFA - nontraditional feed additive oC – degrees Celsius OLA- oleic acid ot – temperature PAA- palmitic acid PLC - Public Limited Company PQI - protein-quality index PUFA - polyunsaturated fatty acid

QMAFAnM- the quantity of mesophile aerobes and facultative anaerobe microbs RK - Republic of Kazakhstan SFA - saturated fatty acid SRI - Scientific Research Institute SRS - Sanitary Regulations and Standards ST RK- Standard of the Republic of Kazakhstan STA- stearic acid USA- United States of America V – vermiculite WHO - World Health Organization WTO - World Trade Organization М – molar mass % - percent

INTRODUCTION

Topicality. President of the Republic of Kazakhstan N. Nazarbayev noted that large-scale modernization of agriculture is necessary, especially in conditions of growing global demand for agricultural products in the message «Kazakhstan Strategy – 2050» - a new political course of the state» to the people of Kazakhstan. It is also necessary to create a feed base for world-class livestock [1]. The head of the state set the task of introducing a fundamentally new system for managing natural resources, aimed all extracting enterprises to introduce only environmentally friendly production facilities [2]. The current state of food production shows, that in many positions needs of the population in food products are not met [3]. To a greater extent, this applies to livestock products. There is a decrease in the competitiveness of the domestic market for livestock and poultry products, which makes it vulnerable to imports, weakening the food security of the territories. The specific gravity of livestock products is very high in the import of food products. The main reasons for this situation are the weak development and low productivity of the livestock sector [4], which are associated with the lack of full-value feeds. The way out is seen in the complex development of feed production. Many animal feed mills in the course of market reforms actually ceased to exist, and the feed mill industry function inefficiently virtually ceased to exist, and the feed mill industry operates inefficiently [5]. Also, with the decline in volumes, assortment and higher prices for high-protein raw materials in the feed industry in recent years, problems have arisen in the production of high-grade, nutritionally balanced feed [6]. It is necessary to search for new sources of raw materials, create a variety of feed additives based on them and thereby expand the raw material base and improve the quality of mixed feed to solve the problem facing the feed industry. Fodder plays an important role in the diet of animals and birds. Productivity and health of cattle depend not only on the amount of food, its usefulness defined set of essential substances, but also its sanitary quality [7, 8]. The concept of feed quality includes a set of indicators of the initial chemical composition of the feed (moisture content, protein, carbohydrates, fiber, fat, macro- and microelements), and its changes depending on the shelf life. It is in the process of storage may occur the quality of fodder can deteriorate by chemical (peroxide and acid number) and biological (contamination with pathogenic bacteria and fungi) indicators [9]. An important factor in solving all these problems is the use of non-traditional feed additives in rations [10]. Of these numbers the highest value acquires natural minerals - aluminosilicates, which have higher sorption and ion-exchange properties [11]. The economic feasibility of the use of natural minerals in various branches of agriculture is confirmed by many publications [12]. Among these natural aluminosilicates is vermiculite. Due to its physico-chemical, ion-exchange and sorption properties, vermiculite is a biologically active agent for

10 increasing productivity and natural resistance, preventing diseases and toxicoses, and also for improving the quality of the final products of livestock and poultry [13, 14]. However, the information about the effect of Kazakhstan deposits’ vermiculite supplementation as feed additive on poultry growth and productivity are limited. The purpose and research problems. The main purpose of our work was the possibility of using feed additives based on domestic expanded vermiculite in poultry farming and to study their influence on the quality of meat and eggs. To accomplish this goal the following tasks were set: - Investigate the veterinary and sanitary requirements for production of Kulantau deposit`s vermiculite for feed preparation; - To study the effects of different doses of the vermiculite on the laboratory animals; - To study the effects of vermiculite on the quality of fishmeal when it stored; - To study the morphometric parameters of the laying hens` eggs while used feed additives based on vermiculite; - To study the preslaughter and postslaughter indicators of broiler chickens, when used feed additives based on vermiculite in the diets; - To veterinary and sanitary assessment of poultry products and determine the degree of influence of the tested feed additives on the quality and safety of the products; - Determine the content of residues of pesticides in feed and feed additives based on vermiculite. Scientific novelty of the research results. For the first time, vermiculite of domestic production for feed preparation was studied and veterinary and sanitary evaluation was given to it. The optimal dose of vermiculite as a sorbent for fish meal during its storage is established. For the first, the influence of domestic natural aluminosilicate on physiological and biochemical indices of blood, natural resistance, productivity and quality of eggs and shells was studied on laying hens and broiler chickens in the production conditions. The harmlessness of vermiculite confirmed in the experiment by anatomical and morphological and histological studies. The optimum dose of sorbent for laying hens and young broilers is determined, which increases the productivity and natural resistance of the organism. The economic justification of expediency of the vermiculite application in poultry farming is given. Practical value of the work. When adding vermiculite to the fat containing feed, the acid number decreases and remains stable during storage, the shelf life of the product is extended. Substantiated data about positive effect of vermiculite on the technological properties of fish meal, on the physiological state, natural resistance and productivity of laying hens and broiler chickens have been obtained. Biophysical studies of the shell of birds have shown that vermiculite improves the quality of the shell and, accordingly, the violation of eggs decreases. The economic feasibility of using fishmeal with the addition of vermiculite has been confirmed.

The results of experimental studies can be used in the educational process for veterinary-sanitary expertise, morphology, biochemistry. The carried out researches allow to recommend application of vermiculite in poultry farming with the purpose of increase of safety of a livestock of birds, meat efficiency, increase of food and biological value of meat, updating of mineral and protein metabolism in an organism of birds. Research results are used in the education of students of the Kazakh National Agrarian University, Almaty Technological University and the Warmino Mazury University in Olshtyn in Poland (Appendixe A). Approbation of the work. Materials of the thesis and the results of scientific and experimental studies have been presented at international scientific conferences: - XLV Scientific Session of Group of Amimal Nutrition knzia PAN. Olsztyn, Polska, 2016; - The XVII International Academic Congress «History, Problems and Prospects of Development of Modern Civilization», Tokyo, Japan, 2016; - Materials of the International Scientific and Practical Conference "Modern Trends in Science and Education", Krakow, Poland, 2016; - Materials of the International Scientific and Practical Conference "Fundamental and Applied Scientific Research", Ekaterinburg, 2015; - Materials of the XII International Conference "Development of Science in the 21st Century", Kharkov, 2016; - Foundation of the first president of the Republic of Kazakhstan — Elbasy. Council of Young Scientists. X International Scientific Conference "Innovative Development and Demand for Science in Modern Kazakhstan", Almaty, 2016; - 4THVETIstanbulGROUP Congress, Kazakh National agrarian university, Almaty, 2017. Publication of research results. According to the materials of the dissertation published 15 publications, including six in journals recommended HAC ("Research results", "Intellect, idea, innovation", "Vestnik of the Shakarim State University", "Vestnik of Science of Kazakh Agro Technical University named after S. Seifullin"), which reflect the main results of experimental studies, 7 -in the proceedings of international conferences, 1 - in journal with high impact factor «Journal of Elementology» included in the database of Web of Science, 1 – in journal «Journal of Pharmaceutical Sciences and Research», included in the Scopus database. Volume and structure of the dissertation. The thesis work was presented to a common pattern. It consistsof an introduction, literature review, materials and methods, results of the research, discussion of results, conclusions, list of sources used 224 items. The dissertation is presented on 139 pages of text, drawn up in compliance with the required standards, illustrated with 38 tables and 22 figures.

1 REVIEW OF LITERATURE

1.1 Development of poultry farming in Kazakhstan In the Address called "Kazakhstan way-2050: Unified goal, common interests, common future" of the President of the Republic of Kazakhstan N.Nazarbayev to the people of Kazakhstan according to the adopted Concept on the transition to a "green" economy, to 2030 year it is said about the need for the development of agrarian science, about the need to create experimental agrarian and innovative clusters [15]. The development of the poultry industry is economically conditioned, socially beneficial and the most promising direction in ensuring the food safety of the Republic [16]. Today modern management of industrial poultry farming has reached a qualitatively new level [17]. As in the whole world, and in our country, purposeful breeding work is carried out to increase the potential, both in meat and in the egg direction. The bird has the highest effect of converting plant protein to animal protein, favorably differing in this indicator from cattle and pigs. Possessing unique qualities of self-sufficiency, the bird strikes very high indicators of intensification. Conversion of feed is less than two units in the production of one kilogram of egg mass or broiler meat, for production of one kilogram of pork is required - 4 to 5 kg of feed, for production of beef 7 to 10 kg. Indicators of production of food protein in terms of a unit of live weight in laying hens is 8-10 times higher corresponding indicator of cows with milk yield of 8 thousand liters a year [18, 19]. Poultry meat is considered to be one of the most popular and widely distributed food products [20]. The modern, intensive production of poultry products has achieved phenomenal profits in the efficient and economical production of high- quality and safe types of meat, eggs and bio-products [21]. Poultry production will continue to grow all over the world. To 2030 year, developing countries will produce twice as much as developed countries [22, 23]. Similarly, poultry farming has the ability to quickly ensure the production of dietary food, eggs and meat with low cholesterol content. Demand for these foods contributes to rapid development of industrial poultry farming, both in our country and abroad [24, 25]. Egg is a special product. In chicken eggs are collected all the nutrients that are required for a living organism for normal development and functioning. The benefits of eggs are explained by the presence in them of vitamins necessary to man: B, C, D, E, as well as useful amino acids [25]. The protein does not contain fats and cholesterol; therefore it is an ideal food for convalescents after viral, cold and other diseases. The protein of fresh chicken eggs has bactericidal properties, is necessary for hemopoiesis processes, improves the functioning of brain cells, and is a preventive agent for the formation of cataracts. In the chicken egg’s yolk are all fat- soluble and most water-soluble vitamins, the main reserves of minerals. Yolk is rich in lipids, proteins, vitamins, minerals [26] [28]. Chicken egg consists of protein, which is almost containing 100% albumin (ovalbumin), and yolk, which contains 7 different proteins: albumin, ovoglobulin,

13 kalbumin, ovomucoid, ovomycin, lysozin, avidin [28]. Chicken egg’s yolk contains a high amount of fat, but it is mainly polyunsaturated fatty acids and monounsaturated fatty acids [29]. Epidemiological studies show that populations that consume large amounts of omega-3 fatty acids have lower rates of breast, prostate and colon cancer than those that consume fewer omega-3 fatty acids [30, 31]. The amount of saturated and monounsaturated fatty acids in eggs is affected by fats in the feed and feed additives. The content of PUFA in the egg can be changed with the help of dietary supplements [32, 33]. Incipience of poultry farming in Kazakhstan refers to the 1930s, when collective and state farms began to appear here. However, the industry gained the high growth rates only in 1964, after a corresponding government decree and transfer to an industrial basis. Due to the specialization and concentration of production, Kazakhstan poultry farming in a short time has become one of the most powerful in Asia. The 80 poultry farms were built, producing 3.5 billion eggs a year and 150 thousand tons of poultry meat [34]. Currently, there are only 25 poultry farms works in Kazakhstan with a total output of 793 million units of eggs and 150 thousand tons of meat, of which more than 200 million eggs are produced by OJSC "Almaty Kus". This shows significant potential of both production and sales in the Republic of Kazakhstan [35]. Poultry farming is characterized by a positive dynamics of development, due to the significant support of the state; the number of birds is steadily growing. It is planned that by 2020 it will reach 60 million bird [36]. According to the statistical agency of the Republic of Kazakhstan, the production of broiler meat has expanded in Kazakhstan, the number of birds increased in 2006 from 65 thousand tons to the expected 160 thousand tons in 2016, it is also expected to double, reaching 246 thousand tons in 2020 [37, 38]. Despite significant progress in the production of poultry meat and eggs over the past five years, the degree of import dependence on this product remains high (imports of poultry increased by 45%, reaching 192,000 tonnes in 2012, representing 61% of poultry consumption domestic market), which is directly related to the high cost of local products and its low profitability [39, 40]. At the same time, the transformation of the agro-food system of Kazakhstan formed a deficit of production, organizational, financial and innovative resources in the poultry product subcomplex, which significantly limited the possibilities for increasing the production efficiency of high-quality and competitive marketable products. The crisis state of the agro industrial complex after the collapse of the Soviet Union led to a large-scale import technological dependence of poultry farming, which complicates the solution of the problems of food and economic security in the country [41, 42]. Competitiveness of the poultry product subcomplex weakens the underdevelopment of the market for industrial, economic and organizational and institutional infrastructure of the agro-industrial complex; insufficiency and non- compliance with international norms of the regulatory framework of the subcomplex (standards, technical regulations); weak integration between enterprises of the

14 subcomplex and food industry [43, 44]. Economic conditions of management, determining the low efficiency of the economic mechanism of meat and egg poultry, include such features of the poultry and egg products market as the dominance of overseas suppliers in the egg market, significant seasonal fluctuations in domestic demand for eggs, leading to significant losses and losses. The reasons for relatively low prices for the sale of subcomplex products are the import and technological pressure of large international competitors; weak market position of enterprises in relation to retail chains, as well as increased concentration and market power of retailers [45]. The growth in the cost of poultry meat and egg products is determined by the critical import dependence in terms of the logistics of the subcomplex, the growth of tariffs for electricity and other energy carriers. In addition, the stability of the industry is threatened by currency risks on the import component of production. The cost price of products of technologically backward enterprises traditionally remains high due to low payment for feed and high mortality of birds [46, 47]. One of the most important problems to be solved in the coming years is to increase the volume of economically efficient production of poultry meat and high- quality egg products with minimum costs of feed, labor and financial resources in difficult economic conditions [48]. The most important economic task of modern industrial poultry farming is to improve the efficiency of feed use as a factor that has a decisive influence on the realization of the genetic potential of poultry productivity [49]. Feeds and feed additives, as sources of nutrients, minerals and biologically active substances and energy, have a diverse effect on the viability and productivity of the poultry. Poultry industry is one of the main consumers of mixed fodders, the costs for which in the cost of eggs and dietary meat exceed 70% [50]. The overall provision of balanced poultry feed does not fully meet the needs in Kazakhstan. Also notes the lack of conformity of feed quality to the standards. The abolition of centralized provision with feed and the introduction of market prices for them adversely affected on work of poultry farms. Many poultry farms have switched to independent preparation of mixed fodders. Due to the fact that purchased and own preparation of mixed fodders do not fully meet the current level of productivity and genetic potential of the poultry, minimization of costs for the production of poultry products at maximum yield is not ensured. So, the pre-slaughter live weight of the broiler is 1.2-1.8 kg at the consumption of compound feed 2.7-4.3 kg per 1 kg of gain, while in the USA it is not more than 2 kg, and in France - 1.8-2.0 kg. Inadequate balanced of mixed fodders lead to a loss of up to 20% of the gross harvest of eggs and a decrease to 30-35% of meat yield [51, 52]. The problem of supplying the fodder base in the republic's economy is largely solved independently by virtue of its capabilities and components. Taking into account that poultry farming is an industrial sector, it is absolutely necessary to develop the feed base and feed mill industry, which will increase the efficiency of the poultry farms [53]. Today, the state provides the necessary support to poultry farming in the form of subsidizing the production of marketable eggs, it is 20% in the cost of finished products [54, 55]. However, poultry farming should gradually move to independent production without the help of the state and effectively contribute to the

15 replenishment of the state budget. In this regard, there are primary tasks, but further intensification of the industry. It is necessary to improve the system of relationships in the industry and include in it the missing production, namely: to create a breeding and selection center, to establish the production of veterinary drugs and quality cheap feed additives of domestic production [56, 57].

1.2 Veterinary and sanitary examination of poultry products Poultry farming is one of the main branches of the agro-industrial complex providing the population with high-grade food products. Poultry meat is an affordable and dietary source of protein in the human diet [58]. In the last decade, in the context of the general economic crisis, there has been a decline in agricultural production in our country, in particular poultry farming, which is currently unable to fully provide the population with eggs and meat. In this regard, the food market began to be filled with livestock products of imported production, including poultry meat, and this product often does not find a market in its countries due to its poor quality (expired shelf life, the content of banned substances, and etc.) [59]. From meat products, chicken hams are imported into our country in unlimited quantities, because of their relative cheapness, which are the most affordable and consumed meat products for the widest sections of the population [60]. From numerous reports in the mass media it follows that ham imported in large quantities from abroad, in contrast to domestic products, have very low quality indicators, which is due to the use of poultry for growth of growth hormones, biostimulants and other substances, negatively affecting both the health of the bird itself and the health of the consumer of this product - a person. It should be noted that in the producing countries this production is not realized at all, in connection with which the issue of quality control is very important [61, 62]. Concern for human health is now more complex task than ever before. An important part of this work falls on veterinary specialists which carry out measures to improve the quality of livestock products and are responsible for obtaining benign food products harmless to humans [63]. Veterinary and sanitary expertise is one of the branches of veterinary science that studies the methods of sanitary and hygienic research of food products and technical raw materials of animal origin and determines the rules for their veterinary and sanitary assessment [64]. Veterinary and sanitary examination is a scientific discipline that develops research methods and veterinary and sanitary assessment of products of animal origin. Veterinary-sanitary expertise has gone a long way of development. Veterinary and sanitary inspection of meat and pre-slaughter inspection of animals began to be used from the second half of the 17th century [65]. The term veterinary-sanitary expertise was introduced in the 1920 year. The main practical importance of veterinary and sanitary expertise is the prevention of diseases transmitted to humans through food and technical products of animal origin [66]. By the order of the Minister of Agriculture of the Republic of Kazakhstan dated from April 1, 2008 No. 199 (registered in the Register of state registration of

16 regulatory legal acts of the Republic of Kazakhstan on April 28, 2008 under No. 5198), the Rules for conducting veterinary and sanitary expertise of food products to determine its safety [67]. The rules are developed in accordance with the laws of the Republic of Kazakhstan dated from July 10, 2002 "About Veterinary Medicine" and on July 21, 2007 "About Food Safety", establish the procedure for conducting veterinary and sanitary expertise of food products at all stages of its life cycle and apply to all subjects, engaged in the procurement (slaughter) of animals, the production, processing and sale of food products subject to veterinary supervision [68]. Veterinary control is a part of state supervision in the field of agrarian field which has certain specificity in a view of that veterinary is complex of specific science knowledge and practical basis, directed to diseases research and food intoxication of animals their preventive measures, diagnosis, medical treatment and liquidation, the provision of state veterinary-sanitary control institutions compliance with legal requirements in the field of veterinary and also the protection of people against diseases which are common for humans and animals [69]. Under the veterinary and sanitary examination in the Rules means checking the conformity of animals, products and raw materials of animal origin to veterinary standards for food safety indicators by a complex of organoleptic, biochemical, microbiological, parasitological, toxicological and radiological studies in accordance with the procedure established by the authorized state body in the field of veterinary medicine [70]. To assess the quality of meat, it is important to know its biological value, which characterizes the result of interaction between the product and the body. Biological value depends from quality of the protein components, their digestibility, and also the balance of the amino acid composition. It is determined by harmlessness, nutrition, biological activity, organoleptic properties of poultry products [71]. Harmlessness characterizes the absence of specific and nonspecific toxicity (increasing the endogenous decomposition of protein and other substances) of the body, which is important for protecting against the contaminating effect of foreign substances on the poultry organism and the use of various hormonal and non-hormonal stimulants, biological and chemical fodder, and antibiotics [72]. The presence of residual amounts of antibiotics in meat affects the results of bacteriological studies, so their definition is important in terms of hygiene. If in a poultry infected with any microorganisms that have a hygienic value, this or that antibiotic is contained in a sufficient concentration for bacteriostatic effect, then bacteriological examination of such a product can produce negative results, despite the fact that the product does not meet the requirements for organoleptic indices. In the case where the concentration of antibiotic in the product is sufficient to suppress the growth of microorganisms, pathogenic bacteria will be present in them in a very small amount, and mainly individuals with increased resistance. They will multiply when the level of antibiotics is below the minimum bacteriostatic concentration. Consequently, when the results of bacteriological examinations are an incorrect evaluation it is possible to produce low-quality products [73, 74]. Biological expertise

17 allows for the combination of the composition and properties of the product to quickly identify the presence of undesirable, harmful factors. The harmlessness of the product and its nutritional value are interrelated parameters of quality. The meat of a sick bird is 15 to 20% lower in nutrition than the meat of a healthy bird [75]. The chemical composition of poultry meat does not completely determine its biological properties, but it is important for quality assessment, and also determines the nutritional (energy) value [76]. Very important for assessing the quality of products has their organoleptic properties. For the consumer of interest are the color, taste, smell, juiciness and tenderness of meat [77]. The color of meat is due to the presence of coloring agents (myoglobin -90%, hemoglobin - 10%). Oxyhemoglobin is formed (the meat is bright red) when hemoglobin is combined with oxygen, and when decomposed it turns into carboxyhemoglobin (dark red color). Myoglobin passes into methemoglobin when prolonged contact with oxygen. Meat acquires a brown color [78]. The intensity of coloring of meat is influenced by the species, breed, sex, age, mode of fattening of birds, as well as the conditions and duration of meat storage, the depth of maturation processes, pH value [79]. A light red color indicates a well- drained fresh meat. The appearance of green coloration is associated with the formation of sulfomyoglobin as a result of the myoglobin reaction with hydrogen sulphide, which is formed by the decomposition of sulfur-containing proteins by microflora. Taste and smell are the main indicators of meat quality. They are formed due to the content and a certain ratio of extractive substances, which are easily oxidized, not resistant to high temperature and at the same time dramatically change their properties. Taste and smell depend on the age of the bird, sex, the ratio of tissues in the meat. In the meat of a young bird these qualities are less pronounced than in the meat of adult birds [80]. Consistency of meat is closely related to such indicators as tenderness, juiciness, softness [81]. Often the consumer, in evaluating the meat consistency, prefers its smell, taste and color. It is proved that juiciness, tenderness, taste and other commodity and technological properties depend on the moisture- binding ability of meat. Therefore, the significance of this ability of meat in its various state and storage is of practical importance. Meat with a darker color is characterized by greater juiciness and less weight loss during cooking, a high pH, which increases the water binding capacity. At pH 6.8, tenderness becomes most pronounced and has an inverse relationship to the content of connective tissue [82]. For post-mortem veterinary and sanitary examination of bird carcasses, experts should follow the rules of veterinary inspection of slaughter animals and veterinary and sanitary examination of meat and meat products [83]. The bird sent for sale must be examined by a veterinarian in advance. The owner of the bird is obliged to provide a veterinary certificate (form number 1) or a veterinary reference (within the administrative area) with obligatory indication of the data on the well-being of the area for infectious diseases [84]. Poultry carcasses are delivered to the market intact in semi-gutted form. The skin should be cleaned from feathers and hemp, without ruptures; beak, tail and legs - without contamination and blood clots. Together with the carcass, the parenchymal organs (heart, liver, spleen, lungs) are provided for

18 examination. Examination is carried out on the basis of inspection of carcases and internal organs [85]. When examining the head, attention is drawn to the color and size of the scallop and earrings, the state of the eyes and mucous membranes of the mouth, pharynx and larynx. Inspection of the internal organs begins with the heart, since in some infectious diseases (cholera, smallpox, and salmonella), it has characteristic pathological and morphological changes. Then inspect the liver (with a number of infectious diseases it can be changed). Changes in the lungs and trachea are observed in plague, ornithosis, etc. The kidneys, spleen, oviducts, gall bladder are also being examined. When examining the internal organs determine the degree of bleeding carcass, fatness, skin condition, muscle and fat tissue, feel the limbs and joints. Sometimes there is a situation where it is necessary to distinguish the carcasses of birds killed in agonizing condition or killed after death. The corpse skin is purple- red or bluish, the crest and earrings are blue-violet, blood drops appear on the cut of muscles and internal organs, the site of the cut is even, and hypostases are found in the subcutaneous tissue. The meat of a healthy bird has a pH of 6.0-6.4, the sick has a pH of 6.5 or higher. The carcass and internal organs are sent to the veterinary laboratory for bacteriological and biochemical analysis in case of detection of pathological changes in internal organs or on serous and mucous membranes. Sanitary assessment of carcasses and internal organs is carried out according to the current rules, depending on the diagnosis and laboratory tests: esophagus, goiter, cuticle of the muscular stomach, intestines, trachea, spleen, testes, ovaries, gallbladder are utilized [86, 87]. The quality of each batch of poultry meat is also checked at the examination. Poultry meat is divided into meat of young and adult birds depending on the age. Carcasses include chicken carcasses, broiler chickens, ducklings, goslings, turkeys and cesareas with neocosten (cartilaginous) keel of the breast bone, with a non- corrugated beak, with a gentle elastic skin. On the legs of the carcasses of chickens, turkeys and guinea fowls are smooth, closely fitting scales and undeveloped, in the form of tubercles, spurs. To carcass of adult birds carry carcasses of chickens, ducks, geese, turkeys and guinea fowls with ossified (hard) keel of a breast bone and a keratinized beak. Poultry carcasses should be clean, free of feathers, fluff, hemp and hairy feathers, wax (for carcasses of waterfowl birds exposed to waxing), well drained, without cracks, tears, blemishes, bruises, intestinal remains and cloaca. In semi-gutted carcasses, the mouth cavity and the beak must be cleaned of food and blood, nails - from contaminants, calcareous growths. It is allowed: on bird carcasses of the 1st category - single hemp and light abrasions, no more than two skin ruptures up to 1 cm in length (only not in the chest area), slight peeling of the skin epidermis; on carcasses of a bird of II category - an insignificant quantity of hemp and grazes, not more than three skin ruptures up to 2 cm long, each skinning the epidermis of the skin, slightly impairing the presentation of the carcass. Carcasses, corresponding to the condition of the requirements of the I-st category, and the quality of processing - II category, refer to the II.

Not allowed in the realization of carcass of poultry: not fresh, not gutted, not corresponding to the fatness and quality of processing requirements of the standard, twice frozen, damaged by rodents, having defects [88, 89]. One of the most common types of spoilage is putrefactive decomposition of meat under the influence of putrefactive microflora. The depth of putrefaction is usually characterized by the degree of change in its freshness. Usually putrefaction begins in the surface layer of meat under the influence of aerobic microorganisms that get on it from the external environment. It is also possible for bacteria to penetrate deeper into the interlayer of connective tissue, especially near the joints, bones and large blood vessels. Decay of protein occurs at the rotting. The decay proceeds differently depending on the composition of the meat, the external conditions and the type of microorganisms. At a certain stage of putrefaction, meat becomes unsuitable for consumption, which is caused by unsatisfactory organoleptic indicators, the accumulation of toxic products of the vital activity of microorganisms. The freshness of meat is judged by the accumulation in it of the most common products of putrefaction [90]. The condition of the beak, the oral mucosa, the eyeball, the surface of the carcass and the internal adipose tissue, and the coarse-serous serous membrane are determined by external examination. Cut the muscle fibers of the pectoral and hip muscles. To determine the humidity of the muscles, apply the filter paper to the surface of the muscle incision for two seconds [91]. To determine the consistency, lightly press the surface of the carcass in the region of the thoracic and hip muscles, inspect the carcass and follow the surface alignment time. The smell of fat determines. For this purpose not less than 20 g. of internal fatty tissue are ground with scissors, they are heated in a water bath and cooled for 20 minutes to a temperature of 20-25 °C (the smell of the surface of the carcass and the coarse-bellied cavity is determined organoleptically) [92]. To determine the transparency and aroma of the broth, cut out about 70 gram of muscles, grind. A sample of 20 gram is placed in a conical flask with a capacity of 100 ml., 60 ml of distilled water poured, covered with glass and put on for 10 minute to a water bath. The aroma of meat broth is determined during the heating to a temperature of 80-85°C. The degree of transparency is determined visually by examining 20 ml broth, poured into a graduated cylinder with a capacity of 25 ml. with a diameter of 20 mm. Comparing the results of the organoleptic evaluation of the test sample for each indicator with the requirements of the standard, describe the results of the study and conclude on the quality of the meat [93]. The presence of pathogenic and conditionally pathogenic microorganisms in the tissues and organs of poultry is observed in infectious diseases. In a healthy poultry, endogenous intravital contamination of organs and tissues by microorganisms occurs during transport. Poultry (especially waterfowl) before slaughter due to a change in the situation, lack of fodder reduces resistance and observed the seeding of muscles (primarily limbs) with salmonella and other microorganisms that live in the intestine, gall bladder, and egg follicles. During the heat treatment, when the carcasses are immersed in hot water, the circulating water is contaminated with organic substances and microorganisms. Within a few hours of operation, the number of microorganisms

20 in the vats of the scalding vats increases 100 or more times. Often water is contaminated not only with saprophytic, but also pathogenic bacteria. During the removal of plumage, seeding occurs as a result of damage to the skin of carcasses (cuts, scratches, abrasions) through which microbes penetrate into the subcutaneous tissue and muscles [94]. When removing internal organs (evisceration and semi- evisceration), seeding occurs as a result of cuts and ruptures of the intestinal tract. This occurs often with half-evisceration. During the removal of the intestine through the cloaca, the intestine is ruptured, and the internal cavity of the carcass is seeded with microorganisms, including not only saprophytic, but also conditionally pathogenic forms (E.coli, Proteus). There are often meets salmonella and perfringrens [95]. Veterinary and sanitary assessment of quality and compliance with the standard is given on the basis of data of organoleptic evaluation, microscopic analysis of meat and bird fat [96, 96].

1.3 Food and biological value of meat and eggs Morphologically, meat is a compound tissue complex, which includes muscle tissue along with connective tissue structures, fat and bones. The quantitative ratio of the main tissues, included in the composition of meat, depends on the breed, sex, age and fatness of the bird [98]. Poultry meat by its composition is different from animal meat. Muscle tissue is characterized by higher density and finer granularity, with the most developed is the pectoral muscles; their mass is equal to or greater than the mass of other muscles [99]. The color of muscle tissue in land birds is not the same in different parts of the body and is from light pink (pectoral muscles) to dark red (in the thigh area). Their most important function is to enable the provision of energy for a relatively short time. Since glycogen can be rapidly mobilized, these muscles provide maximum activity for a relatively short time [99]. The yield of all edible parts of chicken carcasses ranges from 56 to 65%, including the percentage ratio: muscle tissue 38-42; internal fat - 0,8; skin with subcutaneous fat 7 - 15; bones 12 to 18. White chicken meat contains 0.17-0.98% fat, but rich in creatinine - 0.1-1.10% and soluble nitrogenous substances - 10.42% compared to red (1.38 - 2.99%, respectively; 0.64 - 0.83%, 8.05%). It is easier to digest than red, but less nutritious. Antonov N.A. (1994) notes the difference between the meat of birds - from the meat of slaughtered animals, that the chemical composition contains more proteins, extractives and salts. Muscle fibers and connective tissue are more delicate structure than in large animals. Fat of birds is fusible, contains little saturated fatty acids. Its nutritional value is close to butter. All these features give the meat of birds an exceptional nutritional value [101]. The chemical composition of the meat is complex, it is not the same for the tissues entering it and depends on the age, sex, fatness, nature and manner of fattening the bird. Poultry meat contains the same chemical substances as meat of

21 slaughter animals - water, proteins, fats, minerals, extractives, enzymes. The main and most valuable in the food aspect of meat is muscle tissue [102]. The water in the muscle tissue is in hydrated-bound and free states. The amount of water in meat ranges from 47 to 78%. Carcasses of different fatness have a different amount of water: the fatter the meat, the less water in it. This is due to the fact that the main carrier of water in meat is proteins. There is more moisture in the meat of young bird than in the meat of old birds [103]. Lipids of poultry meat are represented by triglycides, phospholipids, cholesterol. Their ratio depends on the species and almost does not depend on the age and fatness of the bird. Triglycides (actually fats) in duck meat are 98%, geese - 96%, chickens - 90%, broilers - 82%, others - phospholipids (respectively 2, 4.10 and 18%). A feature of the composition of bird fat is a significant content of unsaturated fatty acids (69- 70% of all fatty acids), including polyunsaturated, most of them are palmitic and stearic. Poultry meat contains a large number of irreplaceable linoleic and arachidonic acids - broilers of the 1st category - 2.1% of meat, geese, ducks of the 1st category - 6% of meat, that is 5-20 times more than in beef and lamb. Than older the bird and the higher its fatness, the more its meat contains the essential polyunsaturated fatty acids [104, 105]. Extractive substances (0.9 – 2.1%) are more in the meat of adult birds, therefore from chickens it turns out not boiled broth - they are better used for preparation of second dishes. Extractive substances are more in red meat than in white. Nitrogen extractive substances include carnosine, anserine, carnitine, creatine phosphate, creatine, creatinine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, purine bases, free amino acids, urea and others. One of the main nitrogen extractive substances is carnosine. It helps to increase the production and separation of gastric juice. The composition of nitrogen-free extractive substances includes: glycogen, glucose, hexose phosphates, lactic acid, pyruvic acid and others. The share of glycogen (animal starch) accounts for more than half in the total amount of nitrogen-free extractives. Nitrogenous and nitrogen-free extractives have a beneficial effect on digestive processes, the absorption of food by humans and give it a special flavor and aroma [106]. Mineral substances contains in the bird meat within 1-2%. Mineral substances are represented by many macro- and microelements. The microelements (copper, molybdenum, fluorine, iodine, manganese, cobalt, nickel, etc.) are of great physiological importance in human nutrition, since they are part of hormones, enzymes and other biologically active substances. Birds meat contain vitamins A, B1, B2, PP, traces of vitamin C. Heat treatment of meat partially destroys vitamins: when frying - 10-50%, sterilization of canned food - 10-55%, while cooking - 45-60% [107]. In the meat of birds contain various enzymes. Some of them serve simultaneously as plastic material for constructing tissue, for example, myogen; others participate in the formation of intermediate compounds or accelerate hydrolytic transformations. For example, lipase catalyzes the hydrolysis and synthesis of fats. Oxidation-reduction enzymes, in particular peroxidase and catalase, are of

22 practical importance in determining the freshness of meat and the recognition of meat of dead birds. Peroxidase and catalase are one of the most common enzymes [108]. Proteins are the main part of the organic substances of muscle tissue and its main nutritional value. Their total content is higher than in meat of slaughter animals. There are more full proteins (myosin, actin) and less not full-fledged proteins (collagen, elastin) in the poultry meat than in meat of animals due to low percentage of connective tissue [109]. In poultry meat proteins contains a full set of essential amino acids, and in the meat of broilers and geese of the 2nd category, goslings of the 1st and 2nd categories of fatness, the essential amino acids are in optimal proportions and amounts. Amino acids are the basis of protein synthesis. They come from protein feed and endogenous sources of the poultry organism. In birds the metabolic pathways of amino acids differ quantitatively and qualitatively from those of mammals. In birds, urea production is decreased, raised in arginine, and probably in glycine, serine and the proline in growing chicks [110]. Except the meat direction, there is a powerful egg direction of poultry farming, aimed at obtaining a food egg. The egg contains all nutrients and biologically active substances necessary for life, which are in easily digestible form and in an optimal ratio. The egg is digested by man on 96 - 98% [111]. A benign chicken egg is a high-value dietary food. One egg, according to its nutrition, is equivalent to 40 grams of meat and 120 to 150 grams of milk, provides 4 to 5 percent of the daily need of an adult in proteins, fats and minerals, and 10 to 30 percent in essential vitamins [112]. The egg contains all the substances necessary for the growth and development of the bird in the embryonic period. In general, a hen's egg (with a shell) contains an average 65.6% of water and 34.4% of dry matter, from which organic 68.3%, mineral 31.7%. Egg includes proteins (albumin, globulin, keratin, etc.), lipids (fats, sterols, etc.), carbohydrates and pigments (carotenoids, porphyrins, etc.) from organic substances. From inorganic substances, the egg contains macro- and microelements of more than 30 titles [113]. Dry matter of protein is mainly contains by protein, yolk – by fats, shell – by minerals. Water accounts for 47 - 49% in the yolk, in the protein - up to 88%. The proteins of the eggs, possess appropriate concentration of all the crucial amino acids, are generally 99% digestible, a norm in which all other proteins are usually evaluated [114]. The chemical composition of the egg is shown in Table 1. The egg protein has all the essential amino acids (table 2), which ensures its high usefulness, accepted as the standard. Quantitatively, amino acids such as leucine and isoleucine, glutamic acid, aspartic acid, lysine, arginine, proline, valine predominate (5 to 12%). Methionine, cystine, tryptophan and tyrosine are contained in a relatively small concentration (1 - 2%). The ratio of amino acids in the protein yolk and protein is same. The egg protein contains also lysozyme, which possesses bactericidal properties [114]. The lipids of yolk consist unsaturated fatty acids (oleic, linoleic, luteolenic, arachidonic, etc.). The ratio of unsaturated acids to saturate is 7:3, which causes a low melting point of fats and their high digestibility. The yolk of a chicken egg contains

0.2-0.3 g of cholesterol. This is about a sixth of the amount synthesized by the human body per day. There is a lot of lecithin in the yolk (8.6%), which is the main constituent of phospholipids. Due to the high level of lipids, the energy supply of the yolk reaches 1600 kJ in 100 g. [116]. The content of some mineral elements in the egg is shown in Table 3.

Table 1 - Chemical composition of yolk, protein and egg shell,% (generalized data)

Component Yolk White Egg shell Water 47,0 – 49,0 85,0 – 88,0 1,6 Dry substances 51,0 – 53,0 12,0 – 15,0 98,4 Protein 16,0 – 16,6 10,3 – 11,5 3,3 Lipids 32,0 – 33,0 0,03 0,04 0,03 Carbohydrates 0,6 – 1,0 0,6 – 0,9 – Minerals 1,0 – 1,1 0,5 – 0,6 95,1

Table 2 - Exemplary amino acid composition of chicken`s egg

Amino acid Yolk White Amino acid Yolk White Lysine 5,77 6,46 Threonine 4,28 5,40 Methionine 1,78 1,04 Valine 5,27 4,60 Cystine 1,18 0,91 Glycine 2,64 2,58 Tryptophan 1,11 1,20 Alanin 4,39 4,13 Arginine 5,05 6,14 Aspartic 8,17 9,04 Histidine 1,79 1,62 Glutamic acid 9,61 8,80 Leucine + isoleucine 11,88 11,02 Serin 4,17 4,86 Phenylalanine 3,4 2,82 Proline 5,54 8,80 Tyrosine 1,78 1,99

There are a lot of sodium, sulfur, chlorine, and potassium in the protein; in yolk - phosphorus, sulfur, calcium. A lot of iron and zinc in the yolk. A total of microelements in the egg yolk of the chicken egg is 3.8 mg, and 2.8 mg in the white. Also, the egg contains aluminum, boron, bromine, lead, silicon, titanium, strontium, vanadium, uranium, arsenic, barium, selenium, which act as catalysts for various reactions in the body. One chicken egg satisfies the daily requirement of a person in vitamin A (retinol) by 13-15%, in vitamin D - by 10-40%, riboflavin (B2) by 8-10%, in cyanocobalamin (B12) by 50-100% [117]. However, the rapid growth in the production of food and hatching eggs until recently was not accompanied by sufficient efforts to maintain their quality at the proper level. Concern for the food, biological and commodity value of them temporarily receded into the background in a situation where the question of the number of eggs was paramount. This was not slow to affect the quality of eggs, 24 which deteriorated noticeably. A common cause of deterioration in the quality of eggs was a negative impact on the bird and the demolished egg unusual for extensive poultry farming factors that arose with the rapid intensification of the industry. A significant increase in egg production of the poultry led to a reduction in the timing of egg formation, increased one-sided physiological load on the laying organism, which was mainly reflected in a decrease in shell quality [118].

Table 3 - Mineral composition of egg yolk and white of chicken`s egg, mg in 100 g (generalized data)

Mineral Content Mineral Content substance substance In white In yolk In white In yolk

Calcium 10 136 Copper 0,0052 0,139 Phosphorus 27 542 Iodine 0,007 0,023 Sodium 189 51 Manganese 0,003 0,037 Potassium 152 129 Cobalt 0,0005 0,023 Magnesium 9 15 Zinc 0,231 3,105 Chlorine 172 147 Molybdenum 0,004 0,012 Sulfur 187 170 Chromium 0,003 0,008 Iron 0,150 6,700

Veterinary and sanitary expertise of eggs is based on GOSTs of the Soviet period, international standards. Basically, all methods of investigating the quality of food eggs can be divided into two groups - without disrupting the integrity of the eggshell and if it becomes necessary to study the contents of the egg, then the eggshell is broken. A great contribution to the study of food eggs made by Professor Tsarenko P.P. [119], who developed a number of unique biophysical methods for determining the quality of eggs.

1.4 Mineral feed additives and using them in poultry farming The most important problem of the country's food safety is providing the population with meat and meat products. An important role in this task is assigned to poultry farming, since in livestock it is the most dynamic and knowledge-intensive industry, capable of overcoming all difficulties in a shorter time and developing steadily [120]. Growth of consumption of agricultural products requires an increase in productivity and a reduction in the cost of production, which can be achieved through the rational use of feed additives in livestock and poultry [121]. Veterinary dietology is the most developed in the countries of the European Economic Community. Kazakhstan in this respect makes the first steps in the use of non-traditional feed additives based on such natural minerals as zeolites, bentonites, schungites and vermiculites [122].

The silicate minerals such as zeolite, shungite and vermiculite are found to be effective as non-toxic, cheap, ecologically advantageous and affordable materials based on their high-sorption capacity and ion exchanges properties. So they are widely used in many fields of industry, agriculture, environment protection, sanitation, veterinary medicine, and animal nutrition. In the poultry industry, feed additives and antibiotics have been used worldwide more than 50 years to enhance growth performance as well as to prevent infection of pathogens and disease [123]. Production technology, experimental studies and implementation of feed additives for livestock and poultry farming on the basis of natural minerals are relevant and contribute to sustainable development of the agricultury [124]. Among the regional, natural aluminosilicates refers vermiculite which has a high adsorption, cation exchange, and catalytic properties [125]. Poultry industry has been looking for improvement of production indexes and broiler growth through breeding changes in detriment of the final quality of products. Many factors such as mineral diet of poultry may lead to alterations in meat quality [126]. A balanced diet is necessary for optimal poultry production [127]. In particular, it has been suggested that additives play an essential role in maintaining the health of poultry [128]. Mineral elements are part of the body, mainly as a structural material, participate in the processes of digesting nutrients and isolating the products of metabolism [129]. In nature, minerals have always been used by wild animals and birds when the body is depleted, after the disease, in the process of bearing offspring. They found in nature aluminosilicates and ate them. Therefore, use of various natural minerals in physiologically tolerated doses is fully justified [130, 131]. Mineral and vitamin supplements in the total feed costs account for only 5-7%, but the efficiency is increased by 10-25% feed consumption per unit product is reduced by 8-15% and the morbidity and die-off is reduced by 20-40 percent [132]. Common feed additives used in poultry diets include antimicrobials, antioxidants, emulsifiers, binders, pH control agents and enzymes [133]. To ensure normal life and high productivity, the bird should receive with the diet mineral substances that play a major role in the metabolism and are the plastic material of the skeleton [134]. Feature of the body of birds, in contrast to other mammals is a high degree of mineral metabolism (including phosphorus-calcium). Deficiency of these substances causes diseases of the musculoskeletal system, decrease in the quantity and biological value of the products. Therefore, poultry farms tend to use regional natural mineral resources that are rich in macro-microelements and promote better digestion and assimilation of essential nutrients in the gastrointestinal tract of birds [135]. Absence or lack of individual mineral elements, as well as a violation of their ratio in the diet leads to a decrease in the effectiveness of the use of nutrients and, consequently, to a decrease in the productivity of livestock. Their deficiency is compensated by natural minerals and premixes in the composition of mixed fodders [136].

The use of mineral supplementation has been an important part of the feed industry. And minerals are typically classified as macro- or micro-minerals, depends on the levels needed in the diet [137]. The problem of mineral nutrition for animal and poultry productionis solved by the use of complete feed and various additives. It is advisable to pay attention to some of the natural minerals, which have a wide range of properties [138, 139]. In search of effective methods to increase the safety and productivity of poultry, the study was aimed at finding all sorts of little-known feed additives taking into account the use of locally available, cheap raw materials. In recent years, the attention of researchers is directed to use natural mineral additives in poultry feeding. To increase the productivity of poultry farming and reduce costs per unit of production, the stimulants of natural origin are increasingly being used [140, 140]. Additives and premixes are not always fully suitable, to optimize the rations for a complex of nutrients and the lack of antimycotoxic activity. In this regard, it is worthwhile to pay attention to some natural minerals having a wide range of properties and whose actions have been revealed in recent years [142]. Recently, domestic scientists have begun to use in feeding animals and birds non-traditional feed additives based on zeolite, bentonite, schungite and vermiculite [143]. In Central Asia and Kazakhstan, deposits of vermiculites, zeolites, bentonites and schungites began to be explored and developed since 1990. In Kyrgyzstan, bentonite-like clays of Nookat, Serafimovsky, and Enilk deposits, palygorskits - 26 deposits, kaolins - deposits of Sogut, Chokr-Bulak, Tossor, Sulukta and others have been identified and recommended to the national economy. But they are not used for the needs of the feed and medical industry and in agriculture [Ошибка! Источник ссылки не найден.Ошибка! Источник ссылки не найден.]. The unconventional mineral feed additives e.g. zeolites, vermiculites and bentonite would increase the efficiency of feed utilization and strengthen the food base. In diets containing fishmeal, meat and bone meal, supplementation in inorganic vermiculite improve growth performance and health of birds; reduce toxicant residues and also production costs [145].

1.5 Vermiculite-its composition and influence to the organism of animals and birds One of the most promising natural minerals, suitable for use in agriculture, is vermiculite [146]. Vermiculite is a silty mineral that is a product of weathering or hydrothermal decomposition of biotite, flogopite, some chlorites and other silicates rich in magnesium [147, 148]. The vermiculite got its name from Latin word ―worm-like‖due to the fact that the available water in it turning into vapor when heated and expanded the finest vermiculite leaves in a direction perpendicular to the cleavage, forming a ―harmonica‖ - highly elongated worm-like crystals [149]. Vermiculite is a siliat mineral that is obtained from volcanic magma resources. High heat treatment creates an expantion in volume, an increase in permeability and a descrease in weight. The obtained product

27 is very light and sterile. With thermal insulated and fire-resistant features, vermiculite is used as the land regulator in agriculture. The chemical composition of vermiculite is: SiO238-46%, AL2O310-17%, MgO 16-35%, CaO 1-5%, K2O 1-6%, Fe2O36-13%, TiO21-3% and H2O 8-16%. Material has a relatively high water-holding capacity (200-325% of weight and 20-50% by volume) and thermal conductivity (0.065-0.062 watt) and it has a gold colored, accordion shaped physical appearance. Although it has the same function as perlite, vermiculite has better thermal properties and lesser dust ratio than perlite [150]. The chemical formula of the vermiculite is depending on the deposit of origin. The elements: K, Na, Ca, Ti and Cr may be present in small amounts. Vermiculite subjected to heat treatment already can be used with the big economic benefit in the various purposes. Indeed, vermiculite doesn’t burn, doesn't break up. It is chemically inert and possesses bio-firmness. It is also ecologically pure; has beautiful gold color [151, 152]. Vermiculite does not contain carcinogenicor harmful impurities to health human and animal. The presence of macro and microelements in vermiculite compositionin a sufficiently large amount distinguishes it from other natural minerals. Vermiculite was discovered in the beginning of the XIX century. Industrial application was received only after 100 years. For the development of several technologies of its application, Yakub Achtyamo received a prize in 1979 of the USSR Council of Ministers for "Research, development of technology and introduction of vermiculite and products based on it on the national economy" [153]. At present, the largest deposits of vermiculite are found in forty countries of the world (USA, Japan, Italy, Canada, Bulgaria, Hungary, etc.). There are deposits of vermiculite in many parts of the world, but only a limited number of sources have industrial development. Large deposits of vermiculite are discovered in central Asia. Also, the basic deposits of vermiculite are concentrated in the USA, the South African republic, Russia and other countries such as the Republic of Kazakhstan [154]. The Republic of Kazakhstan is one of the biggest sources of vermiculite in the word and ten thousand tons annually is produced. Vermiculite is a new material for the Kazakhstan, although it is used in other countries. Due to its physico-chemical, ion-exchange and sorption properties, vermiculite is a biologically active agent for increasing productivity and natural resistance, preventing diseases and toxicoses, and improving the quality of the products of poultry farming. Possessing a high capacity for liquid substrates, vermiculite retains its bulk properties. This allows the preparation of bulk concentrate containing vermiculite, which can be impregnated with various feed additives, vitamins, probiotics and medicines and contain up to 70% of the mass of the liquid ingredient: (fat, vitamins and other medicaments). Many experiments have shown that feeding of this feed additive into basal diet of will result in a significant increase in meat productivity and improved product quality [155]. Natural origin, the ability to prolong the effect of the complex microelement preparations used, can reduce the amount of substances used for the prevention and treatment of productive animals. And the body is enriched with the necessary micro- and macroelements, at the same time, endo- and exotoxins are eliminated from the

28 body and the digestive tract is prepared for better assimilation of the necessary substances. Vermiculite improves digestion by increasing the area of biochemical reactions in the intestine, sorption of low molecular weight metabolites [156]. Vermiculite has been widely added to animal feed for poultry feeding to improve growth performance and health, to reduce toxic residues and to minimize costs of production [157]. Vermiculite is used as a carrier of liquid nutrients due to its high absorbent properties. It is also used as a carrier of vitamins, molasses, choline chloride and other medicinal substances on a liquid basis. High efficiency is achieved when using vermiculite in poultry farming. It has been revealed that when the fraction of vermiculite is added to the mixed fodder, less than 3 mm to 5% by weight, the volume of food consumed sharply increases and promotes an increase in the biophysical properties and the improvement of the chemical composition of the eggs. In eggs, the relative protein mass, protein and yolk indices, vitamin B1 and B2 content increase. Improves the quality of the shell, increases its thickness and reduces the battle of eggs. A particle of vermiculite gives the appeal of food due to a bright shiny surface [158]. Being an environmentally friendly and sterile material that does not contain toxic and heavy metals, it is not a favorable environment for insects and rodents [159]. Vermiculite is added to animal feed, and is also used as a litter on poultry farms. Only the US annually buys about 58 thousand tons of vermiculite ore, of which one third goes to livestock [160]. In Europe, expanded vermiculite is used for coating sugar beet seeds, as this improves their germination capacity and increases yield by 20%. The shelf life and action of vermiculite is unlimited. It does not rot, does not spread mold, it does not contain rodents and insects [161]. It is established that the inclusion of vermiculite in a bird's diet has a positive effect on immunobiological reactivity, protein and mineral metabolism, productivity and safety. The use of vermiculite in the diet of laying hens has a pronounced effect on the physical parameters of eggs: an increase in mass by 2.5%, a thickening of the shell by 10%, egg density increases by 11.3%. The natural mineral vermiculite has sorption, catalyzing and ion-exchange properties, which excludes the accumulation of heavy metals and arsenic in poultry slaughter products [162]. It is concluded that vermiculite has been added to feed for broilers to improve growth performance and health, to reduce toxicant residues and also production costs, based on the description above [163]. The addition to the main ration of 1-5% allows to reduce the costs of concentrated fodders, to improve their structural composition, enriching them with macro and microelements, increasing the flowability of feeds and their eating, and at the same time increasing the egg-laying by 1-3%, egg harvesting by 4% and safety of livestock by 1.5%. The inclusion of vermiculite in the diet of laying hens is accompanied by an increase in the live weight of the bird by 3.4%, a reduction in the pre-laying period by 6 days. The use of vermiculite has a pronounced effect on the commodity and technological qualities of the products obtained. The weight of gutted carcass of experienced chickens increases by 3.3%, the mass of pectoral muscles by 5.2%, the muscles of the thigh and lower leg by 3.9% [164].

In the research Safiullina G.Ya. [164] the enrichment of rations of broiler ducklings from 7-day old age with vemiculite in an amount of 3.0% of the dry matter norm caused an increase in the increase in live weight by 8.9%, the keeping of livestock by 2%. The information about the effects of dietary combination of Kazakhstan deposits’ vermiculite on quality performance and chemical composition of egg in poultry is limited. The technology of production, experimental research and the introduction of bioactive feed additives for livestock and poultry, based on vermiculites, are relevant and contribute to the sustainable development of the agro-industrial sector [166, 167].

2 OWN RESEARCHES

2.1 Materials and methods The experimental part of the work was carried out in between 2014 – 2017 at the departments "Veterinary-sanitary examination and hygiene", "Biological safety", in the Kazakhstan-Japan Innovation Center of Kazakh National Agrarian University, at the Polish Institute of Plant Protection (Bialystok) and in the laboratories of the University of Warmia-Mazury (Olsztyn, Poland), in LLP "Sary Bulak". In accordance with the solution of specific problems, the research scheme looked like a different search for optimal solutions (Table 4). Feeding of broiler chickens and laying hens. The subject of the research were hens "Hisex White" breed and broilers the "Arbor Acres" breed, which were randomly allocated to ten-tier battery birds of 20 birds each, under conventional conditions of ventilation, temperature (17-19°C) and lighting (16 h light d-1). Birds were kept in isolated sections on deep litter with a partial mesh floor. Layer hens were grown from 120 days old for two months, broilers from daily to 42 days of age. All birds had a free access to diets and water and fed with a standard industrial diet of the LLP "Saru bulak". The protocol for this study was approved by the local Animal Experimentation Ethics Committee (Appendixe B). For experimental studies used expanded vermiculite M-150 from Kulantau deposit, fraction 0,5-3,0 mm. Vermiculite of this deposit had a high content of macro and microelements, as previously described above (Appendixe C). Also, vermiculite with fish meal was used as a feed additive in a ratio of 30:70, which showed good results and highly effective during storage compared with other relations. Feeding of the poultry was carried out by dry full-feed compound feed according to the PC recipe, which consists of the following ingredients (%): for laying hens (PK-1-1) - wheat-50, corn-22, soybean meal-1, sunflower meal-12, prelac-1, yeast feed 5, limestone flour-1, meat-and-bone meal -6.5, premixes 1-2. For the feeding of broiler chickens were used the starting (PK-5) and finishing (PK-6) variants of mixed fodders which are presented in table 5. Feed mixtures contained the same components, the only difference was that the mixtures designed for the experimental groups were supplemented with vermiculite: (A experimental group) a basal diet (BD) without vermiculite (V), (B) 97% of BD supplemented 3% V, (C) BD supplemented with 5% V, (D) 97% BD+3% V+ fish meal (1% V and 2% FM) and (E) 95% basal diet and 5% V+ fish meal (1,5% V and 3,5% FM). The research scheme of use the feed additives are given in table 6.

Table 4 - Scheme of the study

1 STAGE OF WORK

Veterinary and sanitary requirements Study of the safety indicators of to vermiculite for feed preparation: vermiculite: - technological properties and fodder - toxicity on laboratory of animals; value of vermiculite from Kulantau - pesticide residues in feed; deposite; - the amount of heavy metals. - mineral composition and microstructure of vermiculite. Effect of vermiculite on the quality of fishmeal when stored. Investigation of the quality parameters of feed additives. Formulation of fodder additives.

2 STAGE OF WORK

The study of the efficacy of vermiculite and feed additives based on vermiculite in the feeding of broiler chickens and laying hens Zootechnical indicators: Meat production of carcasses: - safety of livestock; - determination of fatness; - average daily growth; - weight of gutted carcass; - biometric parameters of eggs. - meat production. Hematologic and biochemical indices of the blood of laying hens and broiler chickens

3 STAGE OF WORK

Veterinary and sanitary assessment the quality and safety of meat and eggs Organoleptic evaluation of meat, Physical and chemical parameters of broth and eggs: meat and eggs -flavor, taste; -pH of eggs and meat to peroxidase; -transparency, richness; -reaction with Nessler reagent; -tenderness. -reaction with sulfuric copper. Chemical composition of meat and Microbiological indicators of meat egg and eggs: -protein, fat, moisture, dry matter; - QMAFAnM - vitamins and minerals; - CFU -amino acid composition; - Salmonella and Proteus -fatty acid composition.

Table 5 - Composition of the diets for broiler chickens

Groups Item Starter feed Grower feed (0 - 21 days) (22 - 42 days) Ingredient, % Wheat 32.80 40.25 Fodder corn 25.00 21.00 Barley 14.30 9.50 Soybean meal 8.50 11.83 Sunflower meal 4.50 4.30 Meatbonemeal 5.00 5.00 Fodder yeast 5.00 5.00 Crystal lysine 0.38 0.38 Methionine 0.32 0.32 L-threonine 0.12 0.12 salt 0.19 0.19 Sodium sulfate 0.16 0.16 Mineral premix1 0.50 0.50 Vit. premix2 0.30 0.30 Others 3.00 1.02 Total 100 100 Note: 1Mineral premix supplied the following as milligram/kilogram of complete feed: zinc, 50; copper, 12; iodine, 0,3; cobalt, 0,2; iron, 100; selenium 0,1; manganese, 110. 2Vitamin premix supplied the following per kilogram of complete feed: vitamin A, 12 IU; vitamin D3, 2500 IU; vitamin E, 30 IU; vitamin K3, 2 mg; thiamine, 2.25 mg; riboflavin, 7.5 mg; pyridoxine, 3.5 mg; cobalamine, 0.02 mg; niacin, 45 mg; D-pantothenic acid, 12.5 mg; biotin, 0.125 mg; folic acid, 1.5 mg.

Study of the mineral composition and microstructure of vermiculite from Kulantau deposit. The mineral composition and microstructure of vermiculite was determined in the Kazakh-Japan Innovation Center using a scanning electron microscope JSM-6510LA. Miscibility, caking and flowability were determined visually under diffuse light. For quality control, 200g samples were taken from the batch. They were combined, mixed and an average sample weighing 1 kg was obtained. The sample was placed in two glass jars, on which labels were labeled: the name of the product and the sample number; name of the manufacturer's company; lot number, release date. Appearance and color of the samples were determined visually under natural light. A sample of a product weighing 200 g, taken from the combined sample, was scattered in a thin layer on a sheet of paper or a clean surface and mixed with a glass

33 rod. Determination the odor – 5 g of the sample was placed in a flask with the capacity of 200 cm3 and 100 cm3 of distilled water was added, intensively boiled in a water bath for one minute. The cooled liquid was vigorously stirred and then allowed to stand for 1 to 2 minutes. Decanted water was organoleptically checked for odor.

Table 6 - Scheme of the feeding experiment of birds

№ Group Conditions Laying hens I A (control) 100% BD B (experimental) 97% BD + 3% V C (experimental) 95% BD + 5% V D (experimental) 97% BD + 3% V+FM (1%V+2% FM) E (experimental) 95% BD + 5% V+FM (1,5%V+3,5% FM) Broiler chickens I I A (control) 100% BD B (experimental) 97% BD + 3% V C (experimental) 95% BD + 5% V D (experimental) 97% BD + 3% V+FM (1%V+2% FM) E (experimental) 95% BD + 5% V+FM (1,5%V+3,5% FM) Abbreviation: BD – the basal diet, V–vermiculite, FM – fishmeal

For the determine the mass fraction of moisture, a sample weighing 5 g was placed in a previously dried to a constant mass and a weighed bag and dried in a drying oven at a temperature of 105-100 °C for 30 minutes until constant weight. The metal pails with a sample were weighed. Then it was further dried for 15 minutes and weighed again. The determination was repeated until the difference of the last two weighings was no more than 0.02 g. The metal pails with a sample were cooled in a desiccator with anhydrous calcium chloride or sulfuric acid before each weighing. The mass fraction of moisture was determined in parallel in two samples. The mass fraction of moisture (W) in percent was calculated by the formula: m – m х 100 W= 1 2 m1 – m (2), where: m1 - is the weight of the pail with the sample before drying, g; m2 - the mass of the pails with a sample after drying, g; m - the mass of the empty pails, g. The arithmetic mean of the two parallel determinations was taken as the final result of the test. Veterinary and toxicological evaluation of vermiculite. The experiment was conducted at the Vivarium of the Veterinary Clinic of the Kazakh National Agrarian University.For studies were taken 25 white mice with average weigh 14-16 g. They were divided into five groups with five animals in the each (three males and two 34 females). Mice of the first, the second, the third and the fourth groups were fed by basal feed with addition of the vermiculite in doses 1; 3; 5 and 7% respectively for two weeks. Animals of the fifth group were fed only by the basal diet and they served as the control group. During the experiment the survival rate, the weight, the absolute growth and the average daily weight gain of animals, hematological parameters of blood were determined by conventional veterinary methods. Blood tests were carried out at the end of the experience. Determination of water-soluble toxic substances of the vermiculite was performed by Kuznetsov Toxicity Test Method [168] on the protozoa Paramecium caudatum. In the experiment was used the culture of P.caudatum, received from the Almaty Filial of the Republic Veterinary Laboratory. At the first step of the experiment the samples of the Kulantau deposit’s mineral were placed into standard glass tubes. Then the samples were poured by distilled water in proportions of 10:90; 30:70 and 50:50. After this the tubes were shaken in the shuttle machines for 2-3 hours and the tubes stood for 24 hours at a temperature 4-10°C. Then, for studying of toxicity, two drops of the prepared extract were taken and transferred by graduated pipettes to a glass slide and one drop of the cultural medium with 40 exemplars of P.caudatum was added. After this the slide glass was placed in a Petri dish with filter paper moistened with water. The criterion for determining the sensitivity of protozoa was the time from the beginning of the exposure test extract to death ciliates (stop motion and decay). The observation was carried out for two hours. Determination of the quality of fishmeal when stored with vermiculite. The subjects were: fish meal with 20% fat content, from the sanitary-veterinary utilization plant in Almaty and expanded (particle size 0.5-3.0 mm.) vermiculite from the Kazakhstan deposit obtained from the processing enterprise of LLP "AVENUE". To study the effect of the mineral vermiculite on the safety of the product, we were laid in different concentrations of the additive for storage with vermiculite and without the addition of vermiculite (table 7).

Table 7 - Scheme of the experiment

Samples Ratio of the samples Sample 1 Vermiculite (100%), without Fish meal Sample 2 Fish meal (70%) + Vermiculite (30%) Sample 3 Fish meal (80%) + Vermiculite (20%) Sample 4 Fish meal (90%) + Vermiculite (10%) Sample 5 Fish meal (95%) + Vermiculite (5%) Sample 6 Fish meal (97%) + Vermiculite (3%) Sample 7 Fish meal (99%) + Vermiculite (1%) Sample 8 Fish meal (100%), without Vermiculite

The samples were put in special bags in accordance with GOST 13502-86 and stored in a dark room. The room temperature was t = +15 ºС, humidity φ = 55-60%. Each month, samples were taken from each pie and examined the physical and 35 chemical properties of these samples and gave a veterinary sanitary assessment to them. The duration of the study was 6 months. In the work used conventional and special methods of researching raw materials and finished products. Determination of the quality of fishmeal was carried out in accordance with GOST 2116-2000 «Meal from fish, marine mammals, crustaceous and invertebrates. Specifications». Total contamination of microflora was determined in accordance with GOST 25311-82. Determinations of the acid number of fat were carried out in accordance with GOST 13496.18-85, acidity - according to GOST 13496.12-98, the amount of moisture - according to GOST 13496.3-92. Determination of crude protein by the Kjeldahl method GOST 13496.4. Color, odor, appearance were determined organoleptically. Miscibility, caking and flowability were determined visually. We used special clamps to determine the granulometric composition. We did manual work in lab lighting. To determine the efficacy of the sieve, two standard slicers have been used: a solid grid №20 with a hollow opening of 1.8 mm and a hole with diameters of 2 mm in diameter of 1.8 mm. We used special balls made of natural and artificial rubber to accelerate. Evaluation of the results of experimental studies was carried out using modern methods of calculating the statistical validity of measurement results with the help of software packages of Microsoft Office Excel 2007 and Statistika 6.0 for Windows. Determination of residual amounts of pesticides in feed and feed additives for birds. Five samples were examined, of which 2 were the main rations for laying hens (PK1) and broilers (PK5), 1 vermiculite, 1 fish meal, 1 feed additive (vermiculite 30% + fish meal 70%). All reagents used were of analytical grade. To prepare the sample, the QuEChERS method was used, because the method is simple, cheap and effective [169 - 171]. The pesticide standards were purchased from Dr. Ehrenstorfer Laboratory (Augsburg, Germany). The purities of the standard pesticides ranged from 98,5% to 99,8%. The internal standard isoproturon-d6 obtained from Sigma-Aldrich (Steinheim, Germany). Formic acid, acetic acid, ammonium formate and ammonium acetate were purchased from Merck (Darmstadt, Germany). HPLC and LC-MS grade acetonitrile and methanol were purchased from POCh (Gliwice, Poland). LC-grade water (18 MΩ cm) was obtained from a MilliQ water purification system (Millipore Ltd., Bedford, MA, USA). Magnesium sulfate, sodium chloride, sodium citrate dibasic sesquihydrate, sodium citrate tribasic dehydrate were purchased from Agilent Technologies (Santa Clara, USA). C18 was obtained from J.T. Baker (Deventer, Holland). Chitin and diatomaceous earth were purchased from Sigma-Aldrich (Steinheim, Germany). Stock solutions of pesticides (around 1000 µg mL-1) were prepared separately by dissolving an accurately weighed amount of each reference standard in acetone. The combined working standard solutions were generated by serial dilution of the stock solutions with the same solvent. The working standard solutions were used for the preparation of matrix-matched standards within the concentration range of 0.005–2.0 µg mL-1 and for the spiking of samples in the validation studies. The internal standard

(IS), isoproturon-d6 solutions was prepared in the same way as described above. All the stock and working standard solutions and IS was stored in freezer at about –20°C until analysis. Five grams of sample was weighed into a 50 mL disposable polypropylene centrifuge tube and 10 mL of water was added. Next, 100 mkL 5 μg mL-1 internal standard solution (isoproturon-d6) was added followed by 10 mL 1% formic acid in acetonitrile. The tubes were immediately shaken vigorously for 1 min and vortexed for 1 min using a digital Vortex-Mixer (Velp Scientifica, Usmate, Italy). Then the extract was stored at -20 °C for 15 min. Then 4 g anhydrous magnesium sulfate, 1 g sodium chloride, 1 g trisodium citrate dihydrate and 0.5 g disodium hydrogen citrate. The tubes were immediately shaken for 1 min, vortexed in a Vortex-Mixer for 1 min and then centrifuged in a Hettich Cetrifuge (Rotina 420, Germany) for 5 min at 4500 rpm. Acetonitrile extract was transferred into a 15 mL tubes and the samples were stored at -60 °C for 30 min. Then 6 mL aliquot of the acetonintrile supernatant was transferred to a polypropylene centrifuge tube containing 150 mg anhydrous magnesium sulphate, 25 mg PSA and 2.5 g GCB. The tubes were vortexed for 1 min and centrifuged at 4500 rpm for 10 min. One ml of the final extract was filtered through a 0.45 mkm hydrophilic PTFE filter, transferred into the appropriately labeled autosampler vial and subsequently analyzed via LC–MS/MS An Eksigent Ultra LC-100 (Eksigent Technologies, Dublin, CA, USA) liquid chromatography system was operated at a flow rate of 0.35 mL min-1 without split using a SunFire C18 2.5 mkm, 2.1 x 75 mm (Waters) analytical column, maintained at 40°C during the experiments. The volume injected into the LC-MS/MS system was 10 mkL. The binary mobile phase consisted of water with 0.5% formic acid and 2 mM ammonium formate (phase A) and methanol with 0.5% formic acid and 2 mM ammonium formate (phase B). The gradient elution starting at 95% A and 5% B was held for 1.5 min, rising linearly to 10% A and 90% B in 2,5 min and was held for 2.5 min. After ramping, the mobile phase composition was returned to the initial condition in 1 min, and this was held for 4 min for re-equilibration. System MS/MS 6500 QTRAP (AB Sciex Instruments, Foster City, CA) was used for mass spectrometric analysis, equipped with an electrospray ionization source (ESI). The capillary voltage was maintained at 5000 V for positive ion mode and the temperature of the turbo heaters was set at 450°C. As the nebulizer gas (GS1), auxiliary gas (GS2) and curtain gas (CUR) the nitrogen was used at a pressure of 55, 45 and 35 psi respectively. As the nebulizer and collision gas nitrogen was used, too. Optimization of the compounds was performed by injecting individual standard solutions directly into the source (flow injection analysis methods - FIA). Postslaughter examination of carcasses and organs. Veterinary and sanitary examination of slaughter products was carried out according to the current GOST. The subjects of the study were broilers of the same batch; the conditions of their maintenance were the same with observance of optimal parameters of the microclimate. The following were used GOSTs: GOST 18292-85 «Slaughter poultry. Specifications», GOST 7702.0-74 «Poultry meat. Methods of sampling. Organoleptic

37 methods of quality assessment», GOST 31962-2013 «Chicken meat (carcasses of chickens, broiler-chickens and their parts). Specifications». After slaughtering examination of poultry products has features related to the anatomical structure of the poultry and processing technology. The bird lacks lymph nodes, the serous membranes of the medullary cavity are poorly accessible for examination, the lungs and kidneys are located in the depressions of the skeleton and in semi-exhalation only the surface of the carcass and the intestine are available for examination. The study of carcasses and organs was carried out in the following order: they began with external examination of the carcass, determined the correctness of slaughter, the degree of bleeding, and the presence of pathological changes on the skin and in the joints. Consistency was determined by lightly pressing the finger on the surface of the carcass in the region of the thoracic and hip muscles, where a fovea formed and followed the time of its alignment. When examining the head and neck, attention was drawn to the condition of the crest, earrings, earlobes, sinuses, beak, mouth and eyes. On the skin of the head and neck, note the presence of lesions, determine the color of the comb and earrings. When examining the beak, attention was paid to color, glossiness, dryness, elasticity. In the oral cavity, the condition of the mucous membrane of the mouth, tongue, throat and pharyngeal area (color, odor, mucus, nodules, films, caseous plugs) was determined. When examining the eyes, the condition of the cornea was determined: transparency, convexity, paleness, size of the eyeball, presence of mucus, swelling of the supraorbital cavity. With external examination of the head and neck, the presence of signs characteristic of smallpox, diphtheria, plague, cholera, paratyphoid, laryngotracheitis, conjunctivitis, scab and other infectious diseases was determined. Inspection of the internal organs began with the intestines and mesentery. Then, in the process of complete evisceration, the liver, ovaries, testes, stomach, spleen, heart, kidneys and lungs were examined. When examining the internal organs on the mesentery and in the intestine, the presence of hemorrhages, inflammatory phenomena, fibrin, parasites, helminths, nodules, ulcers and other pathoanatomical changes characteristic of such infectious diseases as plague, cholera, paratyphoid, tuberculosis, mycoplasmosis, leukemia, etc., was determined. When examining the heart, attention was paid to the condition of the heart shroud, its color, the presence of hemorrhages, fluids (its number, transparency). When examining the muscles of the heart - for hemorrhages, nodules and consistency (dense, flabby). When examining the liver and spleen, their size, consistency, color, the presence of nodules, foci of necrosis, hemorrhages, and the nature of the incision were determined. In the muscular and glandular stomach, the presence of hemorrhages (especially on their border), mucus, ulceration, the nature of the contents was determined. When examining the chest cavity, the condition of serous membranes, lungs, kidneys, ovaries and testes was examined. Were determined color, the presence of hemorrhages, exudates, fibrin deposits; the condition of the lungs and kidneys (color, size, consistency, the presence of nodules and other changes). The sanitary well-being and suitability of carcasses for food was judged by the results of post-slaughter inspection.

Organoleptic methods of evaluation. Organoleptic indicators of broiler meat are presented in the form of an act of tasting. The organoleptic method of estimating meat (organ + Greek leptikos is able to perceive), the quality of meat evaluated with the help of the senses [172]. Two carcasses from each group were evaluated. An odor, consistency (only meat), taste, clarity (broth only) and an additional taste were determined organoleptically. The results of the organoleptic evaluation of meat and broth (in points) were reflected in the tasting sheets. The following equipment and materials were used for the test: a household grinder with a bore diameter of 3 to 4,5 mm, GOST 4025 - 95, laboratory scales, a water electric bath, scissors, a scalpel, tweezers, a knife, a conical type flask KN - 100 in accordance with GOST 25336-82, glass funnels of type B in accordance with GOST 25336-82, cylinders with a capacity of 22, 100 cm3 in accordance with GOST 1770-74, glasses of chemical type VN-50 according to GOST 25336-82, glass hour, glass sticks, paper filtering according to GOST 12026 - 76, household gauze according to GOST 11109 - 90. For the tasting, whole carcasses were taken after evisceration or separate parts from the same anatomical areas. Prior to heat treatment, the samples were stored for 24 hours in an open container at 4 °C. When preparing samples for analysis, their taste and odor did not change, they had the same dimensions and cuts, the same temperature, the duration of cooking, the degree of grinding, etc. Samples of meat consumed in hot form, tasted at a temperature of 55-60 °C. When poultry meat was tasted, pectoral muscles were evaluated separately. Samples were numbered and denoted by the letters according to the code, known only to the person responsible for their preparation. Determination of the transparency and aroma of the broth was carried out as follows: a 70g muscle of the breast was excised with the scalpel to the full depth of the muscle and, without mixing them by the samples, was ground twice in a meat grinder. Minced meat obtained from each sample was thoroughly mixed. For the preparation of meat broth, 20g of ground meat, weighed with an error of not more than ± 0,001 g, was placed in a conical flask with the capacity of 100cm3 and filled with 60 cm3 of distilled water. The contents of the flask were thoroughly mixed, the flask was covered with a watch glass and placed in a boiling water bath for 10 minutes. The aroma of the meat broth was determined during the heating to a temperature of 80 – 85 °C by sensing the aroma of the vapors emerging from the slightly opened flask. The degree of transparency of the broth was established visually by examining 20cm3 of broth, poured into a measuring cylinder with a capacity of 25 cm3, with a diameter of 20mm. To prepare the meat broth, the meat samples were carefully washed in water at room temperature and left on a lattice baking tray for 5-10 minutes to drain water. We took at least two carcasses from each experimental group. Samples were weighed, recorded, and then placed in an enamel saucepan, poured cold water in a ratio of 1:2 and immediately added salt at a rate of 1% to the weight of meat, boiled with the lid closed to avoid evaporation of volatile aromatic substances. Immediately after boiling,

39 periodically, foam was removed from the broth surface to prevent the formation of turbidity and small flakes. Meat is considered ready if a colorless liquid flows out when it is pierced with a fork. The approximate time for broiler meat cooking is 30 minutes at a temperature of 100 °C. After the end of cooking the meat was taken out, the broth was allowed to settle and at a temperature of 55-60 °C it was served for tasting in glasses in 35- 40 ml portions. For organoleptic assessment of taste qualities of poultry meat, tasting of broth, cooked according to individual taste indices, was carried out on a five-point scale. To determine the smell of fat, take at least 20 g of internal fat. The smell of internal fat was determined organoleptically with the stirring of its clean glass rod. The smell of the surface of the carcass and the coarse-voiced cavity was determined organoleptically. To determine the smell of deep layers with a clean knife, a cut of muscles was made. Particular attention was paid to the smell of layers of muscle tissue adjacent to the bones. Cooked meat was evaluated for such indicators as tenderness, juiciness, taste and aroma. The quality of the meat broth was evaluated according to the following parameters: taste, aroma, richness, color and transparency. Tenderness (stiffness) is characterized by looseness, softness, structure. The notion of "tenderness" includes: a) ease of chewing; b) the ease with which the meat breaks into parts, that is, its friability and looseness; c) value of the residue after chewing. Friability and friability, basically, reflect the resistance of the muscle fiber to fracture, perpendicular to its axis, and the residue after chewing characterizes the content of connective tissue. When heat-treated meat is softening of connective tissue, mainly collagen, and muscle fibers acquire stiffness. Juicy is a quality characterized by the sensation of meat juice during chewing and abundant salivation. Juiciness is due to the release of meat juices during chewing and the stimulating effect of fat on the secretion of saliva. This relationship exists between juiciness and the content of fat in meat, especially intramuscular, an inverse correlation - between juiciness and loss of meat juices during cooking. Tenderness and juiciness are closely related: the tenderer the meat, the faster the juice is released and saliva is secreted during chewing. The richness, color and transparency of the broth are determined by the sensation of the concentrated meat taste and depend on the amount of nitrogenous and nitrogen-free substances transferred to the solution [173]. In determining the transparency, the nature of the fat spots was taken into account. The taste is composed of flavor and taste. The taste is mainly due to water- soluble components: nitrogenous extractives, glutamic acid, volatile fatty acids, and products of interaction of proteins and carbohydrates during heat treatment. When tasting the qualitative indicators of poultry meat, the main attention was paid to boiled meat, as in this culinary product taste and aroma, as well as tenderness and juiciness are most fully manifested, which can not be estimated in broth. Methods of biochemical research. The biochemical parameters determined were: pH (potentiometric method using the device - universal ionomer I-500), reaction with copper sulfate (CuSO4), with hydrogen sulphide, peroxidase, amino- nitrogen and ammonium reaction by Nessler method.

Determination of the concentration of hydrogen ions produced in the extract were from prepation in a ratio of minced meat and water 1:10 at 15-minute extraction.The pH in the extract was determined. For the detection of the primary products of protein breakdown reaction with copper sulphate was used. A sample of 20 g of minced meat was to added 60 ml of distilled water in a 100 ml Erlenmeyer flask. The flask was covered with a glass and heated in a boiling water bath for 10min. Hot broth was filtered through a padof cotton wool (0,5 cm) in a test tube, and placed in a glass of cold water.In the presence of cereal protein the filtrate was passed through filter paper. A two ml portion of the filtrate was poured into a vialto which was added 3 drops of a 5% aqueous solution of copper sulphate.The vial was shaken for 2-3 times and allowed to stand for 5min, after which the result of the reaction was recorded. If the broth remained transparent, the meat is regarded as fresh; when turbid the broth meat is considered of questionable freshness. If a gelatinous precipitate is present this is characteristic of stale meat. For determining the presence of hydrogen sulphidea meat sample was added to a small flask at up to 10% of its volume. A strip of filter paper soaked in an alkaline solution of lead acetate was lowered into the flask, ensuring that the strip between the in nersurface of the neckof the flaskand the cotton plug did not touch the meat. After 15 min. the response was recorded. In fresh meat piece the filter paper remains unchanged. When in the presence of borderline stale meat in the filter turns a pale brown colour and more state meat it turns a darker brown. For testing the reaction to peroxidase, to 2 ml of the filtered extract was added five drops of 0.2% alcoholic solution of benzidine, and after shaking, two drops of a 1% solution of hydrogen peroxide were added. If the meat is fresh and from a healthy bird, the liquid is immediately coloured a bluish color (subsequently turning to brown). If the meat is of doub table freshness and substandard, bluish color does not appear. For determining the aminoammoniacal nitrogen, 20g of an average sample of minced meat is added to 100 ml of distilled water and pressed for 15 minutes, shaking every 5min. The infusion was then filtered through filter paper. Next, 10ml of the filtrate, 40 ml of distilled water and 3 drops of a 1% alcoholic solution of phenolphthalein were added into the conical flasks. The contents of one flask were used as a reference background colour. The content of experimental flask was neutralized with 0,1N solution of potassium hydroxide which had a slightly pink colour. In the neutralized extract 10ml of 40% formalin solution was added, a spreviously neutralized to a phenolphthalein until of as lightly pink coloration. The liberation of the carboxyl groups of the mixture became acidic, and pink colour disappeared. The contents of the flask were titrated again with the same solution until slightly alkaline pink coloration was regained. The content of amino-ammonia nitrogen in 10ml of the filtrate meat extract in milligrams was calculated by multiplying the flow of 0.1N potassium hydroxide titrationin the second by a factor of 1.4. If necessary, adjustments were made for the alkali titer. The content of amino-ammonia nitrogen in 10ml of extract from fresh

41 meat does not exceed 1.26 mg, in a meat of suspicious freshness - from 1.27 to 1.69mg, and in stale meat- more than 1.69 mg. For determining the Nessler number 2 ml of the filtrate and 0.5 ml Nessler reagent was poured into a tube, the tube contents were gently shaken and left for five minutes. The fluid was then centrifuged for three minutes and his intensity of its colour compared, on the white background compared, with the colour of liquids on the bichromates cale. Assessment about the freshness of meat: fresh meat up to 1.0 on the Nessler scale; of suspicious freshness 1.2-1.4; stale-1.6 and above. Methods for determining the chemical composition of meat and eggs. The chemical composition of the meat was determined by a set of methods: The moisture content was determined by the gravimetric method (GOST 9793-74), fat by Soxhlet (GOST 23042-86), total protein by a modified method of Kjeldahl (GOST 32008- 2012), minerals by burning in a muffle furnace (AOAC, 1980). The ash by using a muffle furnace by heating at 550°C for eight hours was determined Methods for determining hematological parameters of blood of birds. Blood in a volume of 9 cm3 for hematological and 3 cm3 for biochemical studies was selected from each specimen from the jugular vein in broiler chickens and in laying hens. The samples were collected within 1 minute of capture to ensure that the levels of the monitored parameters were not affected by any stress induced by presampling handling. The heparinized blood was immediately centrifuged at 837 rmp×g at 4°C for 10 minutes, and plasma samples were stored at −80°C in Eppendorf test tubes until the analyses were performed. The samples for hematological examination were collected in tubes with EDTA and analyzed immediately. Selected plasma biochemical parameters (total protein, calcium, and phosphorus, HGB, HCT, RBC and WBC) were measured. Hematological studies were performed on automatic hematology analyzer «MELET SCHLOESING MS4/5» with a veterinary kit (France). After the whole blood was coagulated, the serum was separated and studies were carried out on an open biochemical analyzer BioChem FC-200 (USA) on such parameters as total protein, calcium and phosphorus. Determination of the live weight of birds. The birds were weighed 1, 14, 28 and 42 days of the experiment. Changes in live weight, absolute, average daily and relative growth in broilers were judged from their individual weighing in control and experimental bird cells, and at the end of the experiment, individual weighing of the entire experimental broiler population was carried out. Methods for studying organoleptic properties and biometric parameters of eggs. Veterinary and sanitary assessment of eggs was determined according to GOST 31654-2012 Eggs chicken food. Technical conditions. During the organoleptic study, color, smell, damage, contamination, marbling and pigmentation of the shell, the presence of inclusions (spots) in the egg, the location of the air chamber, and taste were studied. In the pan, poured water, put on a heating appliance, brought to a boil. Simultaneously selected eggs for the study in the amount of 2-10 pieces, depending on the lot size and the quality condition, were placed in a gauze bag, which was lowered into boiling water, but after the heating ceased. Simultaneously, the

42 thermometer was lowered into boiling water by 100 °C. Eggs were kept in water for 7 minutes. After the specified time, the bag was taken out of the hot water, the water was poured and fresh water was poured into the pan with a temperature of 20 °C, and a bag of eggs was put there for 6 minutes. The temperature of the eggs was cooled to 35-40 °C, the egg contents were tested. Eggs were opened from the blunt end and immediately detected the smell of the air chamber, then examined the taste of protein and yolk. The color and structure were determined by breaking the egg into two parts, examining the contents of the egg, paying attention to the homogeneity of the color (light yellow) over the entire mass of the egg, as well as the absence of lumps that are difficult to crush when pressed with fingers and a spatula. The biophysical properties of eggs were studied with techniques and instruments of Tsarenko. The indexomer IM-1 was used for determine the shape of the eggs. The strength of the shell was determined by an elastic deformation device PUD-1. The measurement technique reduces to the following. The egg is placed horizontally on the pins of the device table with a sharp pole to the left and, rotating the drum, lift until it touches the rod, and then until the arrow is set to zero. The rod of the micro indicator, taking over the load, presses the shell with a force of 0.5 kg (4.9 N). If the arrow has passed zero, it is reset to its original position and brought again. Press the button at the base of the device to release the egg from the load. The shell, deformed under the action of the load, is straightened, raises the rod of the micro indicator, and the needle of the device shows the value of the elastic deformation in micrometers (μm). The state of the air chamber and its height, the state and position of the yolk and the integrity of the shell were determined by the transmission of eggs on the ovoscope OH-10 by turning them. The quality of eggs was assessed according to the indicators: the number of units of HAU, and the mass of eggs, protein, yolk, shell - on electronic analytical scales to within 0.1g for 5 contiguous days of each month. The thickness of the shell is a micrometer accurate to 0.01mm in three sections of the egg with the mean value calculated. The density of eggs was measured with a hydrometer in salt solutions. Methods for the determination of fatty acids in meat and eggs. Fatty acid composition of meat was determined by gas chromatograph with a flame-ionization detector. Chromatographic separation was performed using an Agilent Technologies 7890A gas chromatograph with a flame-ionization detector (FID) and a 30 m 0.32 mm internal diameter capillary column. The liquid phase was Supelcowax 10 and the film thickness was 0.25 mm. The conditions of separation were as follows: carrier gas–helium; flow rate-1 ml min; detector temperature 250ºC; injector temperature 230ºC; column at temperature 195ºC. Methyl esters of acids were identified according to their retention times, which were compared with those of the mixture of methyl esters of fatty acids in the standard Supelco 37 Component FAME Mix 10 mg/ml in methylene chloride (varied) was applied [174]. For the calculation of the percentage share of fatty acids the Chemostation computer program was used. The fatty acid content is presented as the relative percentage (% total fatty acids) in meat.

The statistical analysis was performed using SPSS 17. One way ANOVA was used for comparison of mean values. The eggs were manually broken and separated into egg albumen and yolk. Yolk lipids were extracted using a standard procedure according to applying chloroform and methanol (2:1 v/v) [175]. The FA composition of a yolk lipid was determined by a saponification/methylation procedure [176]. To the extracted yolk lipids (100 mg), placed in a screw capped glass tube, 4 cm3 of 2 M NaOH was added and heated on a heating block. After 10 min, 5 cm3 boron trifluoride-methanol complexes were added and samples still heated. Next, 3 cm3 isooctane was added to the boiling mixture and heated 1 min. After removing the flask from the heat source, without allowing the flask to cool, 20 cm3 NaCl solutions was added. Then, 2 cm3 of the upper isooctane layer was transferred into vial and small amount of anhydrous sodium sulfate was added. Chromatographic analysis was performed using an Hewlett-Packard-6890 gas chromatograph with a flame-ionization detector (FID) and Supelcowax 10 capillary column (100 m×0.25 mm). The conditions of separation: carrier gas-helium; flow rate-1,5 ml min-1; detector temperature 250°C; column temperature 60°C increase of 5°C min-1 to 180°C. Methyl esters of acids were identified by retention times and compared with mixture of methyl esters of fatty acids (Supelco 37 Component FAME Mix, 10 mg ml-1 in methylene chloride. Method for determining the amino acid composition. The amino acid composition of the meat and eggs was determined by capillary electrophoresis (Method -04-38-2009). The solutions prepared for analysis are transferred to Eppendorf type tubes, centrifuged for 5 minutes at a speed of 5000 rpm. Prepare the capillary for work, between the analyzes the capillary is washed with a leading electrolyte for at least 3 minutes. For each prepared solution under appropriate conditions, at least two electrophores are recorded. Methods for determining the mineral composition of meat and eggs. The total amount of mineral substances was measured by burning dry sample in a muffle furnace at 800°C. The iron was determined by the atomic-adsorption method, using GOST 26928-86, for calcium and magnesium used GOST R-09-066-02; potassium - GOST 30504-97, calcium - GOST 26570 - 95, phosphorus GOST 26657-97, manganese GOST 27997-88, sodium GOST 30503-97, magnesium GOST 30502-97. To determine the mineral substances, the dry ashing method was used. That is, ashing took place in muffle furnaces at 400-600°C, NH (CO) was used to accelerate the disintegration process. The sample was placed in a flask, filled with oxygen and closed. The combustion process took 3 minutes. The resulting ash, in which the metals are in the oxides, was treated with a solution of HCl (1: 1) to obtain soluble metal chlorides. Methods for determining the content of vitamins in products. To determine the amount of water-soluble vitamins in products, the method of capillary electrophoresis using capillary electrophoresis system "KAPEL-105" (method M 04-41-2005) was used. The contents of vitamin E were determined by the method of high-performance

44 liquid chromatography (GOST 12822-2014). To determine the content of vitamin A, a high-performance liquid chromatography method was used (ISO 14565: 2000). Morphological and histological examination of meat of broiler chickens. For histological examination, thin, thick sections of muscle tissue were used. The material was fixed with 10% formalin, conducted through alcohols and poured into paraffin. Sections 5-7 mkm thick were stained with hematoxylin-eosin. For histological examination of meat, the interstate standard GOST 19496-93 "Meat. Method of histological examination». Preparing a mixture of egg white with glycerol and processing microscope slides. Fresh egg white, egg yolk without admixture were whisked to foam, poured onto a large filter (filter paper) pre-soaked with distilled water and filtered over night. To the filtered protein was added glycerol at a ratio of 2:1, stirred and 0,1g of camphor was added to prevent rotting. The result antmixture was applied to a degreased glass slides triturated using a gauzeswab, and dried over a flame burner. Preparing of eosin solution. A 1% solution of eosin was used. For preparation of eosin solution of distilled water was used in an amount of 100 cm3, to which was added 1 g of water-soluble eosin, which was stirred until completely dissolved. Preparation of Ehrlich hematoxylin. Ehrlich's haematoxylin was prepared by mixing 20 cm3 of a 10% alcoholic solution of, 80 cm3 of 96% alcohol, 100cm3 of glycerol, 100cm3 of distilled water, 10cm3of glacialacetic acid and 3g of potassium alum. The resulting solution was poured into awide-mouthjar, tied with gauze and as left in the light to mature for four weeks. The ripened solution was filtered. For fixing, the samples were placed in a 10% neutral formalin aqueoussolution and were sealed tightly. The fixed samples were then placed in flask and inserted through the glass funnel, which was washed with cold running water for 15 minutes. After completion of preparation of the sample pieces of a cut size of no more than 3cm3, were collected and sealed in gelatin. Well-washed slices were washed in a 12.5% aqueous gelatin solution for six hours and then in a 25% aqueous gelatin solution for 24 hoursin an oven at a temperature of 37°C. The pieces were then laid out in a Petri dish filled with a fresh blocked 25% aqueous gelatin solution and rapidly cooled in the refrigerator. After cooling, the excised blocks were added to a 20% formalin solution for 12 h. Before cutting with the microtome the blocks were washed. From fixed specimens were excised 15x15x4mm size pieces such that it included the outer surface of the primary sample, as well as the underlying layers, to a depth of 15 mm. Slices were placed in a microtome, and frozen sections were prepared in thicknesses from 10 to 30 microns. With a microtome knife finebrush slices were transferred to a petri dish containing tap water. Under intact slices a glass slide treated with egg white and glycerin was quickly introduced. A slice was removed from the water to the middle of the glass by holding it in position with adissecting needle. Then the slice covered with dry filter paper and, by pressing the paper by hand, stuck on to a glass slide. Staining of slices. Sections were first stained with alum hematoxylin Ehrlich for three minutes, and after a further two minutes, were washed in water. To remove

45 excess hematoxylin, slices were added to a 1% solution of hydrochloric acid until a pink colour was attained, then ammonia water until a blue colour was attained, and then washed with water for two minutes. Slices were stained with 1% aqueous eosin for one minute and rinsed with water. There after, slices were placed undera cover slip. Prepared histological preparations were examined under a biological microscope BIOLAMM-1. Cooked preparations were examined and photographed using a microscope «LEICADM4000B» [177].

3 RESEARCH RESULTS

3.1 Veterinary sanitary requirements and technological properties of vermiculite from Kulantau deposit for feed preparation In recent years, the attention of researchers is directed to the use of non- traditional types of feed additives in feeding animals and poultry. Studies carried out by domestic and foreign scientists show that vermiculites can be used in feeding poultry as a mineral supplement, which helps increase the efficiency of feed use, strengthen the fodder base, and, in some cases, increase the productivity of poultry. Therefore, the technology of production and experimental research of functional feed additives for animal husbandry and poultry farming based on vermiculite is relevant and contributes to the sustainable development of the agro-industrial sector. Vermiculite was discovered in the beginning of the XIX century, industrial application was received only after 100 years. Vermiculite was first described in 1824 in Millbury, USA. Currently, the largest deposits of vermiculite are found in forty countries of the world (USA, Japan, Italy, Canada, Bulgaria, Hungary and others.). There are deposits of vermiculite in many parts of the world, but only a limited number of sources have industrial development Large deposits of vermiculite are discovered in Kazakhstan, Kyrgyzstan and Uzbekistan. Vermiculite is a new material for the Kazakhstan market, although it is widely used in other countries. Due to its physico-chemical, ion-exchange and sorption properties, vermiculite is a biologically active agent for increasing productivity and natural resistance, preventing diseases and toxicoses, and improving the quality of the end products of poultry farming. Kazakhstan is rich in large deposits of vermiculite raw materials such as Iirsu, Zhylandy and Kulantau of the South Kazakhstan region (Figure 1). Preliminary results of comparative studies, physical and chemical, technological properties, vermiculite samples from various regions of Kazakhstan, Russia and far abroad have shown that the most optimal parameters for feed preparation are the raw materials of the Kulantau deposit, which reserves amount to more than 3.5 million tons. The economic competitiveness of the Kulantau vermiculite plant is due to the proximity of the resource base and the availability of cheap energy sources; low infrastructure costs and a convenient transportation.

Kulantau Shymkent

Figure 1 - Geographical location of Kulantau deposite

"AVENUE" LLP is engaged in the development of the vermiculite of deposit "Kulantau" in the Tulkubas district of the South Kazakhstan region. The plant has been operating since 2003. The LLP signed a contract with the Ministry of Energy and Mineral Resources to develop the vermiculite of deposit "Kulantau" in October 2006. The plant (Figure 2) to the extraction and processing of natural vermiculite has a complete structure, the cycle begins from the extraction of ore and to the release of vermiculite products.In currently the plant produces 1500 m3 of products per month. In 2008 was built the shop on manufacture of exfoliated vermiculite [176]. An important property of vermiculite which indicates its commercial value is the ability to increase in volume 6 - 8 times when heated above 300 degrees. Exfoliated 48 vermiculite has a number of valuable features, among which the most important include the following: - Low density-80-200 kg/m3; - Low thermal conductivity-0048-0.06 w/m°C; - High sound absorption coefficient at frequency of 1000 Hz-0.7-0.8; - High resistance to fire, melting point-1250°C; - Small thermal expansion coefficient-0.000014; - Non-toxic; - Beautiful golden color; - Low hygroscopicity, durability, it does not decay, eternal; - Application temperature - -260°C - +1200°C.

Figure 2– Plant of vermiculite

b) Figure 3 - Kulantau quarry for vermiculite extraction (a,b)

Figure 4 - "Screening" system with the necessary size of fractions (a,b)

Figure 5 - Bins for finished products

The workshop is equipped with a technology that excludes the possibility of distortion of the native properties of Kulantau vermiculite. Technology of Kulantau plant corresponds to similar production of natural vermiculite in the US, Japan and Western Europe. Kulantau deposit of vermiculites is studied most intensively. Here also pilot-industrial operation of a part of the first phase section, which ensures the testing of vermiculites in the national economy of Kazakhstan [179]. Vermiculite-raw material is a mineral, in the form of a stone of pink-gray color (figure 6), which then converted at the plant into a granular or powdered mineral of different fractions (figure 7).

Figure 6 - Ore of vermiculite

Figure 7 – Expanded vermiculite of the Kulantau deposit

Vermiculite is an environmentally friendly material. It is used in the field of building materials and construction, also in livestock, poultry and agriculture. Vermiculite used as an ingredient in chicken feed. At the same time, the consumption of concentrated feed decreases, the egg-laying rate of hens, the safety of livestock. The addition of mineral sorbent - expanded vermiculite in the amount of about 3% contributes to the correction of feeds: they are enriched with macro- and microelements, some amino acids stabilize, fermentation processes decrease. Increase the hemoglobin content, the number of erythrocytes, the level of protein, calcium, phosphate and other elements in the organism of animals. The expanded vermiculite is used in veterinary medicine as: - the main component of feed; - an inert carrier of fats, vitamins and nutrients in feeds; - a carrier of medicinal products; - a sorption additive in feed for animals and poultry; - a source of microelements; - to improve digestion; - to increase appetite; - for removing radionuclides and heavy metals from the body; - a bedding material for animals and birds, expanded vermiculite saves heat, absorbs moisture and gases, protects the litter from mold and rot; - for incubating eggs; - for germination of seeds for feeding birds. The expanded vermiculite is used in mixed feed: - increases the body's resistance to unfavorable external factors; - increases detoxification functions of the body; - helps to assimilate nutrients from feed; - supports the normal biocenosis of the gastrointestinal tract; - is a source of vital microelements; - increases resistance to stress; - strengthens immunity. Unlike many sorbents, vermiculite is not abrasive and does not damage the mucous membrane of the gastrointestinal tract of adult animals and young animals. Vermiculite - an ideal substrate for egg ripening. In poultry farming, expanded vermiculite is used: - as an additive in feed, - as litter material, - as a substrate in incubators. For veterinary and animal breeding purposes, vermiculite is used, which has a stable chemical composition. For vermiculites that are used as feed additives, the following requirements are put forward. The mass fraction of fluorine, arsenic, lead, mercury, cadmium and benzpyrene is determined when choosing a quarry, once every three months, and also when there are noticeable changes in the physical properties of vermiculite (color, density, viscosity) in the newly developed board.

In nature, vermiculite often neighbors with asbestos. The feature of vermiculite from "Kulantau" deposit is does not contain impurities asbestos (certificate №127 dated 04.06.2009, LLP "PIC Geoanalitika"), which is characteristic of some deposits of vermiculite. Vermiculite be tested for toxicity. The quality of vermiculite is guaranteed by the supplier on the basis of analysis of samples taken from quarries, as well as from a batch of tuff prepared for shipment to the consumer. Vermiculite must also be supplied to livestock farms in paper four-layer bags in accordance with GOST 13502- 86. Each bag must be stamped with an indelible ink or a label affixed with the indication of: the manufacturer; date of manufacture; fractions and brands of vermiculite; batch numbers; number of this standard. When loading and unloading, all precautions must be followed to ensure the preservation of vermiculite. Transportation of vermiculite should be carried out in covered wagons or other covered vehicles. Storage of vermiculite is produced separately by fractions and brands in conditions that do not allow its disintegration, humidification, perspiration and contamination. When storing and transporting, the height of the stack of vermiculite packed in soft containers should not exceed 1.5 m. Substandard mineral supplements enter the mixed fodder markets, which are sold under the brand of vermiculites. The danger for consumption is created by the fact that this product does not meet veterinary and hygienic requirements and its safety is not guaranteed. Therefore, it is necessary for all consumers of fodder mineral supplements to know about the above requirements and adhere to them. Until now, the technological regulations for the production of feed additives based on Kulantau vermiculite have not been developed. Thus, the production technology, experimental research and introduction of bioactive feed additives for poultry farming, based on natural mineral - vermiculite, are relevant and contribute to the sustainable development of the agro-industrial sector.

3.2 Research the mineral components and microstructure of experimental vermiculite The real researches set as the purpose to study the chemical composition of the domestic natural mineral-vermiculite with sizes of particles of 0.6-5.0 mm further used in the form of feed additive for poultry farming. Vermiculites of various fields differ among themselves on the chemical composition [180], therefore we have investigated indicators of the chemical composition of expanded vermiculite (Figure 8). The chemical composition and stereoultrastructure of feed additive were defined on the electronic scanning microscope JSM-6510LA in the Kazakh-Japan Innovation Center (KJIC) of KazNAU. In the picture shown the result of a research of the chemical composition of vermiculite of the Kazakhstan field. At the lower field of pictures parameters of the modes of shooting are specified (on the right: supply voltage, frequency rate of increase, picture scale, number of work and current time).

Figure 8 - Electronic micrography (a,b) and the chemical composition (c) of expanded vermiculite

By electronic and microscopic researches it is revealed that vermiculite generally has the difficult relief of a microsurface formed by the microcrystals and units presented in most cases by fine weight. Units of microcrystals concentrate in the microgeodes and microcracks located in breed rather evenly.

Table 8 - Absorption of fat and water by vermiculite

Vermiculite Weight of Weight after Absorption, g mineral, g absorption, g Absorption of water 25.0 104.5 79.5 Absorption of fat 25.0 77.0 52.0

The color of vermiculite was brown, green-brown, yellow-brown, and gray with a green and silver hue, odorless, appearance-scaly, loose masses, vermicular. The volumetric mass is 123.25 g/l, the humidity is 0.90%, the pH is 7.11. Data to absorption of water and fat is shown in Table 8. Vermiculite of the Kulantau deposit

57 does not contain asbest impurities (certificate №127 dated 04.06.2009, LLP "Geoanalitika PIC", which is characteristic for some vermiculite deposits. The content large of macro and microelements in the composition of vermiculite distinguishes it from other natural minerals. Experimental vermiculite is characterized by a high content of SiO2 (about 17%), Fe2O3 (about 20%). The results of the research showed that vermiculite retains the original component of mineral elements and the prospect of using this product as a feed additive in poultry farming and as a basis for creating veterinary biological products.

3.3 Veterinary and toxicological evaluation of vermiculite The tables 9 and 10 are shown that the investigated vermiculite had not any toxicity signs since all mice remained alive. The difference in weight of the mice at the beginning of experience in all groups was not more than 0.5g.

Table 9 - The results of determining the vermiculite toxicity on white mice

Group’s Mineral dosage, Number of the Among them: № (%) lab mice (n) alive dead 1 1% 5 5 - 2 3% 5 5 - 3 5% 5 5 - 4 7% 5 5 - 5 Control 5 5 -

Table 10 - Weight (g) dynamics of the laboratory animals

Average weight, g Growth Group’s Initial Final Absolute Absolute average Relative № growth, g daily gain, g growth,% 1 14.8±0.1 16.9±0.6 2.1 0.15 14.19 2 15.2±0.6 17.4±0.7* 2.2 0.16 14.47 3 15.1±0.5 17.3±0.6* 2.2 0.16 14.57 4 15.3±0.5 17.4±0.6* 2.1 0.15 13.73 Control 15.1±0.5 17.1±0.8* 2.0 0.14 13.25 * - P<0.05

After 14 days the weight of animals of the second and the fourth test groups noticeably increased in comparison with the control group (P<0.05). The table 10 is shown that the relative growth in the second and the third groups was higher than in the control group too. There was established that all the mice of experimental groups, which diets included the vermiculite, had a healthy appearance, elastic skin, they had a common movable and physiological activities. In these groups the lethality of mice were not established by the end of the experiment. Mice of the control group, what 58 received the same feed without the additives, were healthy, deaths among them were not observed too. The table 11 demonstrated that in the experiment the content of hemoglobin in blood was higher in the groups of mice fed by the vermiculite diet than in the control group of animals. But this indicator had the highest level (more than 11.5g/l in comparison with the control group) in the third group of mice where the diet mineral addition was 5%.

Table 11 - Peripheral blood picture of mice

Parameters Groups of experienced animals 1 2 3 4 Control HGB, g/l 108.3±0.22 109.4±0.21 115.3±0.31* 112.0±0.33 103.8±0.38* MCV,1012l 7.12±0.32* 7.25±0.27* 7.31±0.63* 7.21±0.78 7.01±0.21* WBC,10 9/l 7.28±0.21* 8.38±0.19 7.51±0.33* 7.37±0.43* 8.31±0.17* NOTE: HGB- hemoglobin, MCV- mean cell volume, WBC- white blood cells * - P<0.05

These data shown that the content of erythrocytes and hemoglobin in the blood of experimental mice did not exceed the limits of the physiological norm, but these indicators were more than the control value. The leukocyte formula was within physiological values and differences between experimental and control mice were insignificantly (Appendixe D). Research results what revealed at the table 12 indicated absence of any negative effects of aqueous extracts of different concentrations of the natural mineral to ciliates. At the same time there has been no cessation of movement or changes in the characteristics of the simplest movement (violation of the metachronous rhythm and a spiral trajectory, avoidance reaction at high concentrations in the medium of different chemicals).There were no signs of the beginning and cellular decay. So survival of P.caudatum individual’s was100%.

Table 12 - Results of observation of P.caudatum for 2 hours

The aqueous extract of vermiculite Movement on the control’s background with distilled water (ratio) Movement Cell death Other changes 10:90 Active No No 30:70 Active No No 50:50 Active No No

It is known that determination the LD50 value of the vermiculite in acute experiments was not possible when the toxicological characteristics of this aluminosilicate had been studying. Addition to the diet of the experienced groups’ mice the maximal possible doses of the vermiculite was not caused changes of animals’ physiological functions. The results obtained are similar to those of 59

Hertmann [181]. All animals were mobile and active, ate feed with the additive well, and kept all the reflexes. Physiologically justified that the inclusion of expanded vermiculite into the diet of laboratory animals was accompanied by an increase of mice’s weight. In comparison with the control group a noticeable risen in the weight gain of the animals’ groups fed by diets with 3 and 5% concentration of the additive was established. Furthermore the additional feeding with the vermiculite influenced to growth of hemoglobin and erythrocytes’ concentrations in blood of mice. The cause of this process might be a high content the iron in vermiculite what could be the factor of the hematopoiesis stimulation. The protozoa Paramecium caudatum is widely used as the model object for testing of the natural mineral as a risk factor for animals and birds. This ciliate is characterized by a complex organization: has a high level of protoplasmic differentiation, and in investigations of the substances’ toxicity P.caudatum gives identical results with the data in studies at the multicellular organisms. According to the EU Directive [182], feed for birds is non-toxic if percentage of ciliates’ surviving is not less than 90% after one hour testing of water extracts of the tested substance. In our research the different concentrations of aqueous extracts the Kulantau field’s vermiculite had not demonstrate negative effects to P.caudatum’s movement and survival. So the experienced vermiculite could be classified as non-toxic feed additive for laboratory mice and harmless mineral for P.caudatum.

3.4 The effect of vermiculite on the quality of fishmeal The problematic diet - related period is in the feeding of all kinds of animals and poultry. The ration of the animal is included in the protein feeding habits, which characterized by the high content of fibrous proteins and gentle suppression of hygienic requirements. In the ration is commonly used fish meal. This is feed additive has a high biological value, which confirms the high yield value (more than 85%) and the content of unsaturated amino acids. In addition, it has a very high content of assimilable forms of calcium and phosphorus. And it contains vitamins A, D and B complex. Fishmeal is characterized by a high proportion of indivisible proteins (60- 75%), so this is a very important product for the feed ration of animals and birds. Fodder fishmeal is characterized by a high content of minerals, in particular phosphorus and calcium 5-5.5% and 13%, respectively. In feeds of vegetable origin, they contain no more than 1% [183]. In addition to these positive nutritional properties, fishmeal has certain drawbacks that put its use into question. When fishmeal is stored, the effect of humidity and temperature on the quality of low-fat and fatty fishmeal shows significant changes in the chemical composition and quality of these products. The feed industry receives low-fat (up to 10% fat) and fat (up to 22% fat) fishmeal. At a temperature of 20 °C for 30 days, the amount of crude protein and water-soluble protein in both low-fat and fatty flour with a moisture content of 8-12% decreases. If the storage period is longer, the greater the loss of protein substances. For 250 days the amount of crude protein decreases at a humidity of 8-14% by 3.6-5.4%, and the water-soluble protein by 3.5-5.8% in low-fat flour. In fatty flour - at the same

60 humidity, the loss of crude protein is 4-6%, and water-soluble protein – 10.3-13.2%. Likewise, in low-fat and fatty flour, the amount of ammonia is increasing. When storing raw materials of animal origin the fat fraction is subjected to changes. Oxidation of fish meal fat is one of the most important reasons for its deterioration. The acidic fat number increases by 1.2-1.5 at a humidity of 8-12% for 60 days in low-fat fish meal and peroxide - by 1.4-1.8 times. With an increase in shelf life of up to 250 days, the loss of raw fat was 47%, acid and peroxide numbers increased by 3.6 and 4.5 times compared to the control. In fatty fish meal, the hydrolytic and oxidizing processes proceed even more intensively and the quantitative-qualitative losses increase. Also, a pathogenic microflora can accumulate when storage and transport of fish meal [184]. A promising solution to this problem is to improve the technology of storing fishmeal, which has a high biological value with the use of natural minerals that can optimize the storage quality of this type of raw materials. An important factor in this possibility of using non-traditional feed additives in rations, among which an essential role is played by natural sorbents, in particular vermiculite, possessing valuable properties, rich in mineral composition and widely used in various fields of human activity. Studies have shown the effect of vermiculite on the sanitary and hygienic indices of the quality of the finished product in the process of its storage - total microbial contamination, acidity and humidity. The results of the study of the samples are given in table 13. The pathogenic microflora was not detected when analyzing colonies which grown on nutrient media.

Table 13 - Quantitative content of microorganisms in feed samples

Sample The number of microorganisms in 1 g of feed, CFU/g of feed at the beginning of the after six monthes additive experiment bacteria fungus yeast bacteria fungus yeast 1 2×104 - - 5×103 - - 2 8×104 - - 2.4×105 - - 3 10×104 2×102 - 3.15×105 4×102 - 4 14×104 3×102 - 7.3×105 5×102 - 5 21×104 3×102 - 11.6×105 4×102 - 6 24×104 4×102 - 11.85×105 6×102 - 7 26×104 5×102 - 15.15×105 7×102 - 8 27×104 5×102 - 15.3×105 7×102 -

The microflora of fishmeal was represented mainly by mold fungi of the genus Penicillium, Aspergillus sp. and bacteria - mainly genus streptococci, micrococci, sarcin and non-spore sticks. In sample 2, where used 30% vermiculite the mold of the genus Penicillium, Aspergillus sp. were not detected. The contamination of microorganisms (bacteria)

61 containing 20-30% of vermiculite, decreased immediately after production by 170- 190 thousand microorganisms/g, after 6 months of storage by 121.5-129 thousand microorganisms/g. Table 14 shows the results of the acid number of fat in feed samples. The acid number in the experimental sample 2 (FM-70%, V-30%) was reduced from 17.16 ± 0.21 to 7.94 ± 0.32 or 53.7%. For six months of storage, this indicator was consistently lower by 32.46 or 60.8% of the control value. Thus, can speak of the canned action of vermiculite on animal feed.

Table 14 - Acid number of fat in the samples.

Terms of storage Group Indicators At the beginning After 3 After 6 of the experiment monthes monthes 1 V -100% - - - 2 FM -70%, V -30% 7.94±0.32 10.00±0.45 12.02±0.27 3 FM -80%, V-20% 10.52±0.41 14.99±0.31 18.31±0.51 4 FM -90%, V-10% 13.59±0.43 15.07±0.24 22.09±0.14 5 FM -95%, V-5% 13.62±0.31 16.17±0.21 22.14±0.19 6 FM -97%, V-3% 13.66±0.02 16.96±0.14 22.24±0.21 7 FM -99%, V-1% 15.03±0.41 21.0±0.54 46.39±0.34 8 FM -100% 17.16±0.21 23.38±0.26 53.34±0.42 Note: FM-fish meal, V-vermiculite

Analyzing the data of Table 14, we can conclude that the natural mineral vermiculite affects the safety of raw materials, reducing acidity (Figure 9), this is explained by the adsorption capacity of the mineral. Vermiculite can absorb liquids up to 2-3 times its own weight. Acid number of fat in experimental groups with vermiculite meets the requirements of regulatory documentation.

50 2 40 3 30 4 5 20 6 10 7 0 8 at the begining of the after 3 months after 6 months study

Figure 9 – Changing of acid number in samples

Taking into account the fact that the amount of fatty acids in the poultry feed should not exceed 10-20%, the results of our study show that the quality of fish meal is well preserved with the addition of 10% -30% vermiculite. Sanitary and hygienic parameters of fishmeal with addition of 30% vermiculite were better: by acid number (AN) by 9.22, by total bacterial contamination (TBC) by 38.09%. After six months of storage, the preservative properties of vermiculite were manifested as follows: AN was lower by 32.46 and TBC by 84.3% compared to the control variant. Based on the results of the performed studies, it is proved that the addition of 10-30% of expanded vermiculite positively affects the decrease in the dissemination of microflora and acidic fat content in the test fish fish samples during storage. This allows to increase the shelf life of the feed additive without loss of quality and to increase the homogeneity of the distribution of animal protein in the finished feed, which is especially important in the production of feed for animals and birds. At the beginning of the experiment, the moisture content of fish meal without adding vermiculite was 18%. The results of determining the moisture content are shown in Table 15. In variants 5, 6 and 7 where 10%, 20% and 30% vermiculite were used, the moisture content was 15.9; 15.1 and 13.9%, respectively, this moisture content is lower by 3.03% compared to the control variant.

Table 15 - The content of moisture of options

№ Month of sampling Options At the After After 6 begining 3 monthes monthes 1 FM -100% 18.0±0.21 15.3±0.61 14.6±0.31 2 FM -99%, V-1% 17.8±0.32 15.2±0.42 14.3±0.85 3 FM -97%, V -3% 17.5±0.34 15.1±0.23 14.1±0.46 4 FM -95%, V -5% 16.4±0.41 14.8±0.62 13.8±0.64 5 FM -90%, V -10% 15.9±0.64 13.9±0.21 11.3±0.12 6 FM -80%, V -20% 15.1±0.14 12.4±0.15 10.1±0.45 7 FM -70%, V -30% 13.9±0.23 11.2±0.33 8.02±0.23 8 V -100% 1.10±0.12 0.21±0.24 0.18±0.11

After three months of storage, the content of moisture was 13.9; 12.4 and 11.2%. After six monthes this indicator was 11.3; 10.1 and 8.02%. We observed these figures after each month of storage. According to the literature, with the preservation of fishmeal, its moisture content increases and its composition is disrupted. As a result of the work, it was found that expanded vermiculite has the ability to maintain the amount of moisture in the test samples, without negatively affecting the quality of the product. Determination of crude protein content. According to the above mentioned data, the amount of crude protein decreases even in the first 30 days of fishmeal storage. 63

The results of quantitative determination of the content of crude protein in the samples are shown in Figure 10. According to the results of the study, the protein content of the sample without vermiculite was 60.0%, where 1% vermiculite was used - the content of the protein was 59.39%, in the sample where 3% vermiculite the protein was 58.98%, where 5% vermiculite -57.61% protein , where 10% of vermiculite is 52.97%, in sample where 20 % vermiculite protein was 46.66% and where 30% vermiculite the content of protein was 43.92%. These indicators were 51.1%, 50.93%, 49.5%, 46.71%, 46.12%, 45.45% and 43.5%, respectively, in samples taken after six months. The amount of protein in variant 1 decreased by 14.83% in six months, the protein content in variant 2 was 14.24%, in variant 3 - 16.07%, in variant 4-18.92%, variant 5-12.93 %, in variant 6 by 2.59%, and variant 7 where was used 30% of vermiculite, the protein content decreased by only 0.96%. This natural mineral - vermiculite has the ability to conserve the amount of biological substances contained in fishmeal and maintain the protein content (Appendixe E).

46,66 43,92

52,97 60

57,61

59,39

58,98

V-0% V-1% V-3% V-5% V-10% V-20% V-30%

45,45 43,5

46,12 51,1

46,71 50,93

49,5

V-0% V-1% V-3% V-5% V-10% V-20% V-30%

Figure 10 - The content of crude protein in the samples at the beginning of the study (a) and after six months of storage (b)

Veterinary and sanitary assessment of the quality of samples and determination of flowability. The color of the samples with vermiculite corresponded to brown-yellow, the odor was from fish meal, and the average size of the vermiculite crystals was 0.1 – 0.5 mm. Consistency was dry. It did not dissolve completely in water. Figure 11 shows the images of the sample at the beginning and at the end of the experiment. In the picture A - fish meal that does not contain vermiculite, picture B - a sample of fishmeal with a content of 30% vermiculite at the beginning of the study. The picture A1 is a fishmeal sample that does not contain vermiculite at the end of the study period, and B1 - the state of the stored sample after six months with 30% vermiculite. According the results of storage, the state of fish meal (B1) deteriorated and a bitter smell appeared.

a1)

b1)

Figure 11 - Pictures of samples at the beginning (a and a1- fishmeal) and at the end (b and b1- fishmeal with 30% vermiculite) of the study

The lumps appeared in the fish meal in the process of storage. And the state of the sample with vermiculite was good, dry, not bitter, there were no lumps. These parameters are characterized by the absorbing capacity of vermiculite, reduce the amount of moisture in the feed, reduce its oxidation level and are not a favorable environment for microorganisms. In the strainer the number of residues of variant A was 15%, and the remainder of variant B was 4%. At the end of the storage period, the remainder of variant A1 was 65%, and the remaining amount of variant B1 was about 22%. This is 43% less than in version A1. The addition of vermiculite to mixed fodders or biological additives is an effective and important solution to problems [184]. The quality of the sample without the addition of vermiculite decreased and was lost. In addition, vermiculite has a positive effect on the conservation of fishmeal and enriches it with important macro- and microelements. The composition of the ingredients has increased, and the uniformity has improved. The above information indicates that vermiculite can be used as a basis for biological products in the field of animal nutrition and veterinary medicine for poultry and livestock.

3.5 Study the content of residual amounts of pesticides in feed and feed additives based on vermiculite In modern agriculture to cultivation and storage of plant crops used synthetic pest control agents - pesticides of active development of the chemical industry. Chemicals received the Latin name of pesticides which used for the complete destruction and effective control of microorganisms, animals and plants that may harm the cultivation and storage of the crop. In terms of their application, they can be classified into the following groups: - Herbicides. Are directed on struggle against weeds. - Bactericides. They are used for the extermination of pathogens (bacteria) of agricultural crops. - Nematocide. Are necessary for effective struggle against round worms. - Insecticides. Destroy insects. - Fungicides. Effective against fungal diseases. - Acaricides. Fight mites. - Zoocides. Used for the destruction of pests and others. Most pesticides are most effective if they are in the vapor state. They are sprayed with special equipment [186]. Use of chemical inputs such as pesticides has increased agricultural production and productivity. However, negative externalities from such use have increased too. These externalities include damage to agricultural land, fisheries, fauna and flora. Another major externality is the unintentional destruction of beneficial predators of pests thereby increasing the virulence of many species of agricultural pests. Furthermore, increased mortality and morbidity of humans due to exposure to pesticides are recorded especially in developing countries. The costs from these externalities are large and affect farmers' returns. However, despite these high costs, farmers continue to use pesticides and in most countries in increasing quantities [187].

Many types of pesticides despite the action of certain types of parasites are toxic to animals and humans. The use of these chemicals in the agricultural sector is strictly controlled. Pesticides can enter people's food, feed and additives for animals, accumulate in the soil, penetrate into ground water [187]. All these unpleasant consequences are side effects from the use of pesticides in pest control. Pesticides can be ingested in fodder when spraying fumigants in warehouses where food is stored. The use of pesticides brings many advantages, as the efficiency, profitability of production, the quality of cereals increases, as does the contamination of agricultural products, water, air and soil [189]. In addition, grain for livestock feeding can be contaminated, and therefore pesticides can be consumed by humans through animal feed. In recent years, in Europe, the United States and Canada, more attention has been paid to monitoring the content of pesticide residues in feed for safety reasons [190]. However, systematic monitoring of residual amounts of pesticides is still not carried out in Kazakhstan. Thus, it is difficult to assess the level of food contamination with pesticides and their presence in feed additives poses a danger to human and animal health. In recent years, herbicides of root action - simazine, atrazine and diuron are becoming more common. These drugs have a set of valuable properties of low water solubility, which reduces the risk of washing them to the root system of fruit trees, the lack of selective action on weeds, long-term toxicity in the soil [191]. For human they are harmless. The effectiveness of these drugs increases with increasing soil moisture. Particularly strong is the level of soil moisture on the action of simazine, to a lesser extent - atrazine and other herbicides of this group. The best results are given by their late autumn or early spring application, immediately after the mechanical treatment of the soil. Annual weeds die from small doses of prepaggs for the destruction of rhizome and root-shoot weeds require higher dosages. With a large species diversity of weeds in the garden, simazine is effective at doses of 10.0-12.0 kg per hectare, atrazine – 8.0kg per 1 ha, diuron – 3.0 kg per 1 hectare. Due to its low toxicity, diuron is not dangerous for bees and other useful insects [192]. The number of mites, springtails, millipedes, wireworms and earthworms in the soil under the influence of increased doses of monuronum is temporarily reduced. To fish monuron and diuron are moderately toxic. For monuron, the upper limit of safe concentrations was 20 mg; the toxic effects for perches were 30 mg/l and for roach 80 mg/l. The definition of pesticides in animal feed and human food is not an easy task, which can only be solved through a special accredited research center. The purpose of this study is to measure the level of residues of pesticides present in feed samples and feed additives based on vermiculite produced in Kazakhstan, and the effect of pesticides in feed for human health.

Figure 12 - Samples of feed additives

Insecticides (including organochlorine, organophosphate, pyrethroids, carbamate and others), fungicides, herbicides and acaricides were investigated. The list of pesticides studied in feeds shows in Table 16. The analyzes were carried out at the Polish Scientific Laboratory, which is accredited in accordance with ISO / IEC 17025: 2005. For this purpose, a gas chromatography method with an electronic detector was used. The level of residual amounts of pesticides was assessed relative to the allowable daily dose, the acute reference dose obtained from toxicological studies, the maximum allowable level. Samples of feed additives for birds were studied (Figure 12). Vermiculite is a product of processing of deposits of Kulantau of Southern Kazakhstan. In our studies in the samples, the residual amounts of methyl chlorpyrifos, diazinon, malathion, methyl pyrimiphos (FOS), aldrin, DDT (including metabolites), ά-HCH (group HOS), cypermethrin, delmetrine (pyrethroid group)) were not detected. Only in the samples W/BIA/0002/16 (Vermiculite with fishmeal) W/BIA/0003/16 (Fish meal) diuron was determined in amounts of 0.0024 and 0.0045 mg/kg (Figure 11). But this does not exceed the maximum permissible concentration. There is no data on the toxicity of monuron and diuron for humans. Diuron. (N- 3,4-dichlorophenyl-N, N-dimethylurea). Produced in the form of 80% wettable powder. Systemic drug, enters the plants mainly through the roots. It is used to control annual weeds in cotton crops (0.5-2 kg/ha) by spraying the soil simultaneously with sowing or before emergence of crops. In moist soil, the drug exhibits greater toxicity. In high doses (above 15 kg/ha) can be used as a herbicide of continuous action. Diuron is low-toxic for warm-blooded animals. Acute toxicity (LD50) for white mice with oral administration is 3600 (3400) mg/kg. Tests for irritation and sensitization of the skin with diuron gave a negative result in guinea pigs [193]. Thus, it can be concluded that the detected residual amounts of pesticides in the samples represent a minor danger to consumers (Appendixe F).

The sample of feed additive W_BIA_0002_16

The sample of feed additive W_BIA_0003_16

Figure 13 - Chromatograms of the feed additive samples W_BIA_0002_16 and W_BIA_0003_16

Table 16 - List of pesticides studied in feeds

3-Hydroxycarbofuran 2 Bifenazate 1 Chloroxuron 1 6-Chlor-3-phenylpyridazin-4-ol 1 Bifenazate 2 Chloroxuron 2 6-Chlor-3-phenylpyridazin-4-ol 2 Bifenthrin 1 Chlorsulfuron 1 Acephate 1 Bifenthrin 2 Chlorsulfuron 2 Acephate 2 Bitertanol 1 Chlortoluron 1 Acetamiprid 1 Bitertanol 2 Chlortoluron 2 Acetamiprid 2 Boscalid 1 Chromafenozide 1 Acibenzolar-S-methyl 1 Boscalid 2 Chromafenozide 2 Acibenzolar-S-methyl 2 Bromacil 1 Cinosulfuron 1 Ametryn 1 Bromacil 2 Cinosulfuron 2 Ametryn 2 Bromuconazole_ISO_2 Clofentezine 1 Amidosulfuron 1 Bupirimate 1 Clofentezine 2 Amidosulfuron 2 Bupirimate 2 Clomazone 1 Aminocarb 1 Buprofezin 1 Clomazone 2 Aminocarb 2 Buprofezin 2 Clothianidin 1 Atrazine 1 Butafenacil 1 Clothianidin 2 Atrazine 2 Butafenacil 2 Crimidin 1 Avermectin_B1a 1 Buturon 1 Crimidin 2 Avermectin_B1a 2 Buturon 2 Cyazofamid 1 Azaconazole_ISO_1 1 Cadusafos 1 Cyazofamid 2 Azaconazole_ISO_1 2 Cadusafos 2 Cycloxydim_ISO_1 1 Azaconazole_ISO_2 1 Carbaryl 1 Cycloxydim_ISO_1 2 Azaconazole_ISO_2 2 Carbaryl 2 Cycloxydim_ISO_2 1 Azinphos-ethyl 1 Carbendazim 1 Cycloxydim_ISO_2 2 Azinphos-ethyl 2 Carbendazim 2 Cycluron 1 Azinphos-methyl 1 Carbetamide 1 Cycluron 2 Azinphos-methyl 2 Carbetamide 2 Cymoxanil 1 Azoxystrobin 1 Carbofuran 1 Cymoxanil 2 Azoxystrobin 2 Carbofuran 2 Cyprazine 1 Benalaxyl 1 Carbosulfan 1 Cyprazine 2 Benalaxyl 2 Carbosulfan 2 Cyprodinil 1 Bendiocarb 1 Carboxine 1 Cyprodinil 2 Bendiocarb 2 Carboxine 2 Demeton-S 1 Benfuracarb 1 Carfentrazone-ethyl 1 Demeton-S 2 Benfuracarb 2 Carfentrazone-ethyl 2 Desmedipham 1 Bensulfuron-methyl 1 Chlorbromuron 1 Desmedipham 2 Bensulfuron-methyl 2 Chlorbromuron 2 Diafenthiuron 1 Benthiavalicarb-isopropyl 1 Chlorfluazuron 1 Diafenthiuron 2 Benthiavalicarb-isopropyl 2 Chlorfluazuron 2 Diazinon 1 Benzoximate 1 Chloridazon 1 Diazinon 2

Continuation of the Table 16

Dichlofluanid 2 Diuron_ISO_1 2 Fenbuconazole 2 Dichlorvos 1 Diuron_ISO_2 1 Fenfuram 1 Dichlorvos 2 Diuron_ISO_2 2 Fenfuram 2 Diclobutrazol_ISO_1 1 Epoxiconazole 1 Fenhexamid 1 Diclobutrazol_ISO_1 2 Epoxiconazole 2 Fenhexamid 2 Diclobutrazol_ISO_2 1 EprinomectinB1a 1 Fenitrothion 1 Diclobutrazol_ISO_2 2 EprinomectinB1a 2 Fenitrothion 2 Dicrotophos 1 Etaconazole_ISO_1 1 Fenobucarb 1 Dicrotophos 2 Etaconazole_ISO_1 2 Fenobucarb 2 Diethofencarb 1 Etaconazole_ISO_2 1 Fenoxycarb 1 Diethofencarb 2 Etaconazole_ISO_2 2 Fenoxycarb 2 Difenoconazole 1 Ethiofencarb 1 Fenpropidin 1 Difenoconazole 2 Ethiofencarb 2 Fenpropidin 2 Difenoxuron 1 Ethiofencarb-sulfone 1 Fenpropimorph 1 Difenoxuron 2 Ethiofencarb-sulfone 2 Fenpropimorph 2 Diflufenican 1 Ethiofencarb-sulfoxide 1 Fenpyroximate 1 Diflufenican 2 Ethiofencarb-sulfoxide 2 Fenpyroximate 2 Dimefuron 1 Ethion 1 Fensulfothion-sulfon 1 Dimefuron 2 Ethion 2 Fensulfothion-sulfon 2 Dimethachlor 1 Ethiprole 1 Fenthion_ISO_1 1 Dimethachlor 2 Ethiprole 2 Fenthion_ISO_1 2 Dimethenamide 1 Ethirimol 1 Fenthion_ISO_21 Dimethenamide 2 Ethirimol 2 Fenthion_ISO_2 2 Dimethoate 1 Ethofumesate 1 Fenuron 1 Dimethoate 2 Ethofumesate 2 Fenuron 2 Dimethomorph_ISO_1 1 Ethoprophos 1 Fipronil 1 Dimethomorph_ISO_1 2 Ethoprophos 2 Fipronil 2 Dimethomorph_ISO_2 1 Etofenprox 1 Flazasulfuron 1 Dimethomorph_ISO_2 2 Etofenprox 2 Flazasulfuron 2 Dimoxystrobin 1 Etoxazole 1 Flonicamid 1 Dimoxystrobin 2 Etoxazole 2 Flonicamid 2 Diniconazole 1 Famoxadon 1 Fluazifop 1 Diniconazole 2 Famoxadon 2 Fluazifop 2 Dinotefuran 1 Fenamidone 1 Fluazuron 1 Dinotefuran 2 Fenamidone 2 Fluazuron 2 Dioxacarb 1 Fenamiphos 1 Flubendiamid 1 Dioxacarb 2 Fenamiphos 2 Flubendiamid 2 Disulfoton 1 Fenarimol 1 Fludioxonil 1 Disulfoton 2 Fenarimol 2 Fludioxonil 2 Disulfoton-sulfon 1 Fenazaquin 1 Flufenacet 1 Disulfoton-sulfon 2 Fenazaquin 2 Flufenacet 2 73

Continuation of the Table 16

Flufenoxuron 2 Furathiocarb 2 Isoxaben 2 Flumioxazin 1 Halofenozide 1 Isoxadifen-ethyl 1 Flumioxazin 2 Halofenozide 2 Isoxadifen-ethyl 2 Fluometuron 1 Heptenophos 1 Isoxaflutole 1 Fluometuron 2 Heptenophos 2 Isoxaflutole 2 Fluopicolid 1 Hexaconazole 1 Kresoxim-methyl 1 Fluopicolid 2 Hexaconazole 2 Kresoxim-methyl 2 Fluoroglycofene-ethyl 1 Hexazinone 1 Lenacil 1 Fluoroglycofene-ethyl 2 Hexazinone 2 Lenacil 2 Fluoxastrobin 1 Hexythiazox 1 Linuron 1 Fluoxastrobin 2 Hexythiazox 2 Linuron 2 Fluquinconazole 1 Hydramethylnon 1 Lufenuron 1 Fluquinconazole 2 Hydramethylnon 2 Lufenuron 2 Fluridone 1 Imazalil 1 Malaoxon 1 Fluridone 2 Imazalil 2 Malaoxon 2 Flurochloridone 1 Imibenconazole 1 Mandipropamid 1 Flurochloridone 2 Imibenconazole 2 Mandipropamid 2 Flurtamone 1 Imidacloprid 1 Mecarbam 1 Flurtamone 2 Imidacloprid 2 Mecarbam 2 Flusilazole 1 Indoxacarb 1 Mefenacet 1 Flusilazole 2 Indoxacarb 2 Mefenacet 2 Fluthiacet-methyl 1 Iodosulfuron-methyl 1 Mefenpyr-diethyl 1 Fluthiacet-methyl 2 Iodosulfuron-methyl 2 Mefenpyr-diethyl 2 Flutolanil 1 Ipconazole_ISO_1 1 Mepanipyrim 1 Flutolanil 2 Ipconazole_ISO_1 2 Mepanipyrim 2 Flutriafol 1 Ipconazole_ISO_2 1 Mepronil 1 Flutriafol 2 Ipconazole_ISO_2 2 Mepronil 2 Forchlorfenuron 1 Iprodione 1 Mesosulfuron-methyl 1 Forchlorfenuron 2 Iprodione 2 Mesosulfuron-methyl 2 Formetanate_ISO_1 1 Iprovalicarb 1 Mesotrione 1 Formetanate_ISO_1 2 Iprovalicarb 2 Mesotrione 2 Formetanate_ISO_2 1 Isofenphos-methyl 1 Metaflumizon 1 Formetanate_ISO_2 2 Isofenphos-methyl 2 Metaflumizon 2 Fosthiazate 1 Isoprocarb 1 Metalaxyl 1 Fosthiazate 2 Isoprocarb 2 Metalaxyl 2 Fuberidazole 1 Isoprothiolane 1 Metamitron 1 Fuberidazole 2 Isoprothiolane 2 Metamitron 2 Furalaxyl 1 Isoproturon 1 Metazachlor 1 Furalaxyl 2 Isoproturon 2 Metazachlor 2

Continuation of the Table 16

Metconazole 2 Monocrotophos 1 Penconazole 2 Methabenzthiazuron 1 Monocrotophos 2 Pencycuron 1 Methabenzthiazuron 2 Monolinuron 1 Pencycuron 2 Methacrifos 1 Monolinuron 2 Pendimethalin 1 Methacrifos 2 Monuron 1 Pendimethalin 2 Methfuroxam 1 Monuron 2 Petoxamid 1 Methfuroxam 2 Myclobutanil 1 Petoxamid 2 Methidathion 1 Myclobutanil 2 Phenmedipham 1 Methidathion 2 Naled 1 Phenmedipham 2 Methiocarb 1 Naled 2 Phenthoate_ISO_1 1 Methiocarb 2 Neburon 1 Phenthoate_ISO_1 2 Methiocarb-sulfon 1 Neburon 2 Phenthoate_ISO_2 1 Methiocarb-sulfon 2 Nicosulfuron_ISO_1 1 Phenthoate_ISO_2 2 Methiocarb-sulfoxid 1 Nicosulfuron_ISO_1 2 Phorate 1 Methiocarb-sulfoxid 2 Nicosulfuron_ISO_2 1 Phorate 2 Methomyl 1 Nicosulfuron_ISO_2 2 Phorate-sulfoxid 1 Methomyl 2 Nitenpyram 1 Phorate-sulfoxid 2 Methoprotryne 1 Nitenpyram 2 Phosalon 1 Methoprotryne 2 Norflurazon 1 Phosalon 2 Methoxyfenozide 1 Norflurazon 2 Phosmet 1 Methoxyfenozide 2 Novaluron 1 Phosmet 2 Metobromuron 1 Novaluron 2 Phoxim 1 Metobromuron 2 Omethoate 1 Phoxim 2 Metolcarb 1 Omethoate 2 Picolinafen 1 Metolcarb 2 Oxadixyl 1 Picolinafen 2 Metosulam 1 Oxadixyl 2 Picoxystrobin 1 Metosulam 2 OxamyI 1 Picoxystrobin 2 Metoxuron 1 OxamyI 2 Piperonyl butoxide 1 Metoxuron 2 Oxamyl-oxime 1 Piperonyl butoxide 2 Metrafenone 1 Oxamyl-oxime 2 Pirimicarb 1 Metrafenone 2 Oxycarboxin 1 Pirimicarb 2 Metribuzin 1 Oxycarboxin 2 Pirimicarb-desmethyl 1 Metribuzin 2 Oxydemeton-methyl 1 Pirimicarb-desmethyl 2 Metsulfuron-methyl 1 Oxydemeton-methyl 2 Pirimiphos-ethyl 1 Metsulfuron-methyl 2 Paclobutrazol 1 Pirimiphos-ethyl 2 Mevinphos_ISO_1 1 Paclobutrazol 2 Pirimiphos-methyl_ISO_1 Mevinphos_ISO_1 2 Paraoxon 1 Prochloraz 1 Mevinphos_ISO_2 1 Paraoxon 2 Prochloraz 2 Mevinphos_ISO_2 2 Parathion 1 Procymidone 1 Mexacarbate 1 Parathion 2 Procymidone 2

Continuation of the Table 16

Profoxydim_ISO_1 2 Pyriproxyfen 2 Tebuconazol 2 Profoxydim_ISO_2 1 Qinoxyfen 1 Tebufenozide 1 Profoxydim_ISO_2 2 Qinoxyfen 2 Tebufenozide 2 Promecarb 1 Quinalphos 1 Tebufenpyrad 1 Promecarb 2 Quinalphos 2 Tebufenpyrad 2 Prometon 1 Quinoclamine_ISO_1 1 Tebuthiuron 1 Prometon 2 Quinoclamine_ISO_1 2 Tebuthiuron 2 prometryn 1 Quinoclamine_ISO_2 1 Teflubenzuron 1 prometryn 2 Quinoclamine_ISO_2 2 Teflubenzuron 2 Propamocarb 1 Rimsulfuron 1 Tepraloxydim_ISO_1 1 Propamocarb 2 Rimsulfuron 2 Tepraloxydim_ISO_1 2 Propaquizafop 1 Rotenone 1 Tepraloxydim_ISO_2 1 Propaquizafop 2 Rotenone 2 Tepraloxydim_ISO_2 2 Propargite 1 Secbumeton 1 Terbumeton 1 Propargite 2 Secbumeton 2 Terbumeton 2 Propham 1 Siduron_ISO_1 1 Terbuthylazine 1 Propham 2 Siduron_ISO_1 2 Terbuthylazine 2 Propiconazole 1 Siduron_ISO_2 1 Terbutryne 1 Propiconazole 2 Siduron_ISO_2 2 Terbutryne 2 Propoxur 1 Simazine 1 Tetrachlorvinphos 1 Propoxur 2 Simazine 2 Tetrachlorvinphos 2 Propyzamide 1 Simetryn 1 Tetraconazole 1 Propyzamide 2 Simetryn 2 Tetraconazole 2 Proquinazid 1 Spinosyn_A 1 Tetramethrin_ISO_1 1 Proquinazid 2 Spinosyn_A 2 Tetramethrin_ISO_1 2 Prosulfocarb 1 Spinosyn_D 1 Tetramethrin_ISO_2 1 Prosulfocarb 2 Spinosyn_D 2 Tetramethrin_ISO_2 2 Prosulfuron 1 Spirodiclofen 1 Thiabendazol 1 Prosulfuron 2 Spirodiclofen 2 Thiabendazol 2 Pymetrozine 1 Spirotetramat 1 Thiacloprid 1 Pymetrozine 2 Spirotetramat 2 Thiacloprid 2 Pyracarbolid 1 Spiroxamine_ISO_1 1 Thiamethoxam 1 Pyracarbolid 2 Spiroxamine_ISO_1 2 Thiamethoxam 2 Pyraclostrobin 1 Spiroxamine_ISO_2 1 Thidiazuron 1 Pyraclostrobin 2 Spiroxamine_ISO_2 2 Thidiazuron 2 Pyrazophos 1 Sulfentrazone 1 Thifensulfuron-methyl 1 Pyrazophos 2 Sulfentrazone 2 Thifensulfuron-methyl 2 Pyridalil 1 Sulfometuron-methyl 1 Thiobencarb 1 Pyridalil 2 Sulfometuron-methyl 2 Thiobencarb 2 Pyrimethanil 1 Sulfosulfuron 1 Thiodicarb 1 Pyrimethanil 2 Sulfosulfuron 2 Thiodicarb 2 76

Continuation of the Table 16

Thiofanox-sulfone 1 Triadimefon 1 Triflumuron 1 Thiofanox-sulfone 2 Triadimefon 2 Triflumuron 2 Thiofanox-sulfoxide 1 Triadimenol 1 Triflusulfuron-methyl 1 Thiofanox-sulfoxide 2 Triadimenol 2 Triflusulfuron-methyl 2 Thiophanate-ethyl 1 Triasulfuron 1 Triforine 1 Thiophanate-ethyl 2 Triasulfuron 2 Triforine 2 Thiophanate-methyl 1 Triazophos 1 Trinexapac-ethyl 1 Thiophanate-methyl 2 Triazophos 2 Trinexapac-ethyl 2 Tolclofos-methyl 1 Triazoxide 1 Triticonazole 1 Tolclofos-methyl 2 Triazoxide 2 Triticonazole 2 Tolylfluanid 1 Tribenuron-methyl 1 Uniconazole_ISO_1 1 Tolylfluanid 2 Tribenuron-methyl 2 Uniconazole_ISO_1 2 TralkoxydimE 1 Trifloxystrobin 1 Vamidothion 1 TralkoxydimE 2 Trifloxystrobin 2 Vamidothion 2 TralkoxydimZ 1 Triflumizole 1 Zoxamide 1 TralkoxydimZ 2 Triflumizole 2 Zoxamide 2

3.6 Hematological and biochemical parameters of the blood of birds when using feed additives based on vermiculite The blood is a liquid tissue of the body, which reflected the physiological condition as mirror. It will change the morphological and biochemical composition of the blood while disordersor infectious diseases function of organ. In practice veterinary medicine using biochemical methods frequently [194, 195]. Hemoglobin concentration (Hb), red blood cells (RBC`s) count and white blood cells (WBC`s) count were determined. Hematological testing is one of the methods that can help detect certain changes in health that may not be apparent from physical examination, but which affect, for example, the condition of the birds [196]. Hematologic studies include hematocrit value (HCT) which indicates the ratio between the volume of plasma and blood cells. The value of hematocrit in all studying groups matches to physiological standards.

Table 17 - Haematological and biochemical indices of laying hensand broilers

Groups Indicators Control 1experimental 2 experimental 3 experi-mental 4 experi-mental (A) (B) (C) (D) (E) Laying hens, n=10 (M±m) HGB, g/l 87,0±1,41 92,1±2,01 101,2±1,40 96,0±1,43 110,5±0,11 HCT,% 29,7±0,82 28,6±1,15 32,1±0,16 31,7±0,61 34,2±0,21 RBC,×1012 / l 4,9±0,43 4,1±0,15 5,02±1,22 4,29±0,13 4,21±0,44 WBC,×109 / l 6,5±1,36 12,5±1,22 7,3±0,46 6,4±0,61 5,2±0,23 Calcium,mmol/ l 1,75±0,32 1,81±0,15 2,78±0,51 1,82±0,62 1,81±0,4 Phosphorus,mg /l 1,52±0,21 2,37±0,23 1,54±0,42 1,55±0,34 1,48±0,14 Protein, g / l 35,8±0,42 37,9±0,42 36,7±0,31 38,4±0,27 42,1±0,61 Broilers, n=10 (M±m) HGB, g/l 119,2±0,4 124,0±0,2 124,0±1,2 128,1±0,4 129,0±0,4 HCT,% 36,8±1,81 39,0±1,23 39,0±0,45 37,7±0,60 39,1±0,12 RBC,×1012 / l 4,47±0,15 4,76±1,21 4,15±0,62 4,64±0,44 4,73±0,61 WBC,×109 / l 8,0±0,21 6,9±0,46 7,7±0,15 6,6±0,14 8,5±0,16 Calcium,mmol/ l 1,38±0,62 1,55±1,13 1,57±0,14 1,83±1,62 1,78±0,64 Phosphorus,mg /l 1,32±0,28 1,43±0,51 1,55±0,14 1,56±1,1 1,38±0,31 Protein, g / l 35,3±0,51 37,3±2,02 37,8±1,23 38,2±1,44 39,1±0,14

Hematologic studies include hematocrit value. Hematocrit indicates the ratio between the volume of plasma and blood cells [197]. As shown in Table 17 the hematocrit variations in the mean value were not statistically significant, being for hens: 32.1% for the dose with 5% V, for the group D and E: 31.7% and 34.2%. For broiler chickens: 39.0% and 39.0% for the dose with 3% and 5% V, 37.7% and 39.1% for the dose with 3% and 5% V with FM. The hematocrit (HCT) of laying hens fed with various dose of minerals significantly was higher in all experimental groups than in the control 29.7% except group B (28.6%). The highest increase of HCT of broilers was observed in the group E (39.1). The amount of hemoglobin (HGB) is an indirect indicator of the body's iron saturation. Low hemoglobin content was for the control groups where birds received only standard feed. Hemoglobin showed mean value oscillations rangin 23g from a minimum of 124.0 g/l for the chickens groups B and C to a maximum of 129.0 g/l for the group E and for the laying hens of 92.1 g/l group B to a maximum of 110,5 g/l for the group E, with statistically significant differences. The highest concentration increase showed hens group E (110,5 g/l). The number of red blood cells (WBC) in the groups B, D and E was lower than in control. The mean values of (RBC) ranged from 4.12·1012 l-1 (B) for hens to 5.02·1012 l-1 (C), for broiler chicken from 4.15·1012 l-1 (C) to 4.76·1012 l-1 (B). Data referring to the leukocyte (WBC) parameters development, as presented in Table 17, showed very significant statistical oscillations of the total leukocyte number, for laying hens situated in the range of 6.4·109 l-1 (D) to 12.5·109 l-1 (3% V and 3% V+FM) and control group 6.5·109 l-1. For broiler chickens situated in the range of 6.6·109 l-1 (D) to 8.5·109 l-1 (E). When using natural feed additives or its combination with fishmeal in all experimental groups the amount of protein increased comparing to the control. It was associated with faster metabolism which was confirmed by higher productivity of birds. The values of the total protein content in the most of birds were within the range of 35-42 g l-1[198]. The hens group E had higher protein concentration of 42 g l-1 than the control (35 g l-1), for broilers higher protein concentration is 39.1 g l-1 (E). The increase of calcium was observed in the all experimental groups of hens than control 40%. This was associated with the highest ion-exchange activity of vermiculite. The highest concentration increase of calcium to control group (1.75 mmol l-1 and 1.38 mmol l-1) showed birds group C (2.78 mmol l-1) for laying hens and D (1.83 mmol l-1) for broilers. The amount of phosphorus in the serum egg-laying hens for B (2.37 mg l-1) with to the control group (1.52 mg l-1) has small increase, with except group of hens feeding 5% V (1.48 mg l-1). The amount of protein, WBC, HGT, calcium was higher in the hens group fed with combination of V+FM (Appendixe G).

3.7 Veterinary and sanitary assessment of the quality of meat and eggs when used the feed additives based on vermiculite in the composition

3.7.1 Effect of feed additives on the based of vermiculite on the growth and productivity of broilerchickens There are several measures that can be used to evaluate the performance of a flock of chickens – growth rate, days to market, mortality, and feed efficiency. This results indicated that an average weight of the all experimental groups fed with vermiculite feed additives was higher than the control group (table 18).

Table 18 - Effects of feed additives on growth performance of broiler chickens

Period (d)

Initial weight BWG (g/bird) (g/bird) Groups 1 14 28 42 A (Control) 47,4±1,2 786,73±25,6c 1502,10±46,2 2007,30±65,1c B (3% V) 46,2±1,1 793,71±31,0d 1531,07±59,2a 2158,00±14,0a C (5% V) 44,6±2,6 796,05±24,3d 1529,16±16,4 2204,21±20,8a D (3% V+FM) 47,3±1,3 805,35±15,5a 1638,47±24,2b 2403,31±20,8b E (5% V+FM) 45,4±2,2 812,21±14,3a 1644,30±57,0b 2506,50±53,8a *BWG- body weight gain, a-d Means in the same raw with different superscripts are significantly different at p<0.0

Table 19 - Effects of feed additives on body weight changes of broiler chickens

Groups Growth A D E B (3% V) C (5% V) (Control) (3% V+FM) (5% V+FM) Relative 1959,9 2110,82 2156,61 2356,01 2459,1 growth, g Average 46,66 50,25 51,35 56,09 58,55 daily gain, g Growth rate 42,35 45,72 46,31 50,81 52,88

In an experimental group E, where chickens fed with vermiculite plus fishmeal, the weight gain was more on average 19% than in the control group. Addition of vermiculite and vermiculite plus fishmeal to broiler’s feed had a significant impact on absolute average daily gain (ADG) and relative growth (GR). Table 19 shows that the difference in weight of the broiler chickens at the beginning of experiment in all groups was not more than 0.4g, after 42 days average daily gain ADG of the C group 80 was 51.35g; D 56.09g, E 58.55g while the control ranged from 47.2g to 47.4g. The growth rate in the control group was 42.3g, in the D group 50.81g and was higher about 10.53g than in the control in the E group 52.88g (V+FM). As a result, feed efficiency was much better in the first weeks up to one month (about 75% of body weight) of broiler chicken production. Body weight gain of birds for the first 14 days of age given a diet supplemented with vermiculite with fishmeal was higher than those given only vermiculite. The body weight gain was not affected by the dietary treatments from 28 to 42 days. This observation is in agreement with the results of [199], who observed no differences in body weight gain of broiler chickens supplemented with different natural feed additives as alternatives to antibiotic growth promoters. In this report, the results demonstrated that broiler chickens fed with the 5%V+FM diets had significantly greater body weight, better average daily gain, relative growth gains and growth rate than birds fed a control diet during the experimental period. The maximum values of weight were recorded in broiler chickens of the third and fourth experimental groups – 2397.31-2501.41g, which is 16.1-19.2% (p <0.001) more than in the control (Table 20). The weight of the gutted carcass of broiler chickens of the control group was 1362.31 ± 1.32 g, which is by 6.6% (p <0.05) less than of the first test group, by 10.1% (p <0.001) less than in the chicks of the second experimental group, 16.7% (p <0.001) less than in the poultry of the third test group and 22.1% (p <0.001) less than in the fourth test group. The yield of gutted carcass of broiler chickens fed with feed additives was higher than in control group.

Table 20 - Meat yield of broiler chickens

Indicators, n=25 Group Postslaughter Preslaughter weight , g Slaughter yield, % weight , g A 2002,50±0,31 1362,31±1,32* 68,03±2,31* B 2146,12±2,35* 1459,32±1,32 68,04±0,51 C 2198,21±3,21 1516,41±2,33 68,98±0,41 D 2397,31±0,62 1636,12±2,31 68,25±0,35 E 2501,41±0,41*** 1750,71±0,53*** 70,07±3,21*** * р < 0,05; *** р < 0,001

The results of weighing the internal organs of broiler chickens showed that the values of liver, heart, lung, spleen, muscle and kidney weight in broiler chickens of the control group and the first test group differed insignificantly (table 21). In broiler chickens of the third and fourth experimental groups showed an increase the mass of internal organs where we used vermiculite and fishmeal. 81

Table 21 - Weight of internal organs of broiler chickens

Groups Indicators A B C D E Liver 39,9 ± 0,3 40,2 ± 0,3 42,6± 0,5 42,6 ± 0,56** 42,9 ± 0,45* A heart 9,8 ± 0,1 9,9 ± 0,1 10,9 ± 0,1 10,8 ± 0,31* 10,9 ± 0,41 Lungs 9,7± 0,2 9,7 ± 0,2 11,7± 0,1 11,7 ± 0,16** 11,8 ± 0,21 Spleen 2,3 ± 0,1* 2,4 ± 0,1* 2,7 ± 0,1** 2,7 ± 0,06 2,7 ± 0,13 Muscular 30,3 ± 0,3 30,3 ± 0,2 33,3 ± 0,3 33,3 ± 0,20 33,4 ± 0,21 Stomach Kidneys 4,9± 0,1* 4,9 ± 0,2 5,5 ± 0,1 5,5 ± 0,03** 5,6 ± 0,32* * р < 0,05; ** р < 0,01.

The poultry of the control group by weight of the liver was inferior to the chickens of the third and fourth test groups by 6.3-6.9% (p <0.05-0.01), by mass of the heart - by 9.2-10.0% ( p <0.05-0.001), lungs - by 17.0-17.8% (p <0.01), the spleen - by 14.8-14.9% (p <0,01), the muscular stomach - on 9.0-9.2% (р <0.01- 0.001), kidneys - on 10.9-12.0% (р <0.05-0.01). Thus, feed additives based on vermiculite in the studied doses contribute to improving the growth and development of broiler chickens, increasing their meat production (Appendixe K).

3.7.2 The qualitative characteristics of eggs when used feed additives based on vermiculite Morphometric measures of eggs are the main economically important parameters of poultry production and they are very varied. Age of the hens and diet have a significant effect on the whole egg, white, yolk solids and on yolk: white ratio of eggs [200]. Inthis study, hens diet based on vermiculite feed additive caused a change of morphometric parameters of eggs (Table 22). Intensity of eggs production of laying hens in the experimental group (C) made 77.25%, and in the experimental group (E) of 87.67% against 68.30% in control group. In the end, collecting eggs from D and E was 3 and 5 units more than the control; productivity per hen in the E test group was 52.6 pieces of eggs for two months. This was 22% higher than in the control group. In all tested groups of hens (B-E) fed with vermiculite plus fishmeal productivity of eggs was higher than control group. Hens fed with vermiculite additive (groups B and C) laid 100 eggs more than control group and about 200 eggs more after feeding with vermiculite p lus fishmeal (groups D and E). The solids contents of whole egg, white and yolk varied within a narrow range among egg sizes. Average egg weight was the highest in group E (63.3g). The level of albumen and yolk of egg was also the highest in B-D groups. 82

However, an analysis of indicators that indirectly characterize the strength of the shell (elastic deformation - ED) reliably indicates a high physical quality of the shell in eggs obtained from layers from experimental groups. In control groups the average ED was 26.9 ± 0.91 mkm, and the experiment was in the range of 19.5 ± 1.01 mkm. These indicators are economic factors in production conditions. These facts allow us to regard vermiculite as a preventive measure of preserving the production marriage - the battle of eggs, and increasing the efficiency of production of table eggs. Eggs of hens fed with 5% V (group C) had the highest weight of shell and its thickness and density. Eggshell breaking strength is a key indicator of egg quality. This research has shown that eggshell breaking strength had a little increase in hens diets fed with vermiculite+FM.

3.7.3 Organoleptical and quality parameters of meat and eggs when used feed additives based on vermiculite Organoleptic assessment of meat. Veterinary and sanitary examination of broiler chicken meat showed the following results: organoleptic parameters were assessed by the appearance of the carcass, the state of the muscles on the cut, the meat consistency, the clarity and aroma of the broth. The muscles of the bird are fully developed, the chest is rounded, the bones are unspecified and the formation of subcutaneous fat is not much. The carcasses of broiler chickens of all experimental groups had a dry surface, the color was pale yellow, the beak was shiny, the oral mucosa had a pale pink color, and the inner and subcutaneous fat was pale yellow. The muscles on the incision were slightly wet, leaving no spots on the filter paper. Its smell was characterized by the type of bird. According to the consistency, the meat is elastic, formed when pressing the pit, aligned. There were no scratches on the skin, it was uneven. After a mulberry tarted bouquet of aroma and light.After cooking the meat, the resulting broth is fragrant and transparent. The organoleptic characteristics of the broiler chicken meat, which were fed of vermiculite, corresponded to sanitary requirements. The results of the reactions conducted to determine the quality of meat are shown in Table 23.

Table 22 - Productivity, morphometric and quality parameters of eggs of lying hens fed different levels of vermiculite.

Groups Parameter A B C D E (Control) (3% V) (5% V) (3% V+FM) (5% V+FM) Number of eggs 820 1034 1030 1266 1284 Number of eggs per hen 41 46,35 46,25 52,15 52,6 Hen house eggs production, 68,3 77,25 77,08 86,92 87,67 % Defective eggs 18 6 3 11 7 Egg weight, g 58,07±0,32 58,71±0,13 59,13±0,42 61,25±0,2b 63,31±0,30c Albumen, g 35,31±0,14a 35,58±0,23 35,68±0,14c 37,81±0,31 38,45±0,32a Yolk, g 17,32±0,17a 17,51±0,21b 17,48±0,08 17,96±0,09a 19,33±0,06a Shell, g 5.44±0,18a 5,62±0,16a 5,97±0,23a 5,48±0,22 5,53±0,16b Shell thickness, mm 0.381±0,002c 0,423±0,006 0,453±0,004c 0.392±0.003 0,405±0,004 Elasticity, mkm 26,9±0,91 24,4±1,61 19,5±1,01 26,3±2,20 24,1±0,35 The density of eggs (g cm-3) 2,14±0,30a 2,31±0,30 2,37±0,20a 2,21±0,10b 2,25±0,40b a-d Means in the same raw with different superscripts are significantly different at p<0.05

Table 23 - Change of biochemical parameters of broiler chickens` meat using a feed additives based on vermiculite

Biochemical characteristics A B C D E of meat Samples of meat 1 2 3 4 5 6 7 8 9 10 Determination of sulfuric ------acid Reaction with a 5% solution + + + + + + + + + + of copper sulfate Reaction withNessler's 0,8 0,7 0,6 0,7 0,6 0,6 0,7 0,7 0,6 0,8 reagent Reaction to the peroxidase + + + + + + + + + + Reaction to the reductase ------(according to M. Kondratov) РН 6,9 6,8 6,7 6,6 6,5 6,6 6,7 6,6 6,7 6,9 bacterioscopy (on the 15 13 12 13 11 12 13 13 12 12 surface) Sanitary assessment + + + + + + + + + +

As a result of reductase reactions, all samples were decolorized after 2.5-3.0 hours. That is, all samples of meat were not contaminated with microbes. The pH of the meat of broiler chickens was 6.5-6.9, which indicates the good quality of meat. When reacting to ammonia and ammonium salts with Nessler reagent, the obtained minced meat extract had a greenish-yellow color and retained its transparency. This reaction is based on the formation of a complex salt - iodide dimercurammonium, which has a yellow-orange color. Depending on the amount of ammonia and its salts in the extract of poultry meat, the degree of coloration, as well as the amount of sediment, varies. The acid number of fat was 0.60-0.8 mg KOH. In the peroxidase test, all results were colored blue and after 1 to 2 minutes they were dyed brown, which gave a positive result. Microscopic analysis of the upper layers of the muscle tissue of broiler chickens showed that 11-13 microbial cells (cocci and rods) were established in one field of vision, which indicates the absence of negative influence of vermiculite on the veterinary and sanitary characteristics of meat. When cooking meat broth is transparent, without flakes, flavored, this corresponds to GOST 51944-2002 «Poultry meat. Methods for determination of organoleptic parameters, temperature, mass". To determine the taste qualities of meat samples and broth of experimental broiler chickens an organoleptic evaluation was performed (table 24). Thoracic muscles are a valuable product widely used in cooking. The fried meat of the control group received a score of 4,58 points, while similar indicators of the experimental groups were estimated slightly higher and amounted to – 4.61; 4.61; 4.61; 4.68 and 4.68 points respectively. Also, samples of boiled meat of broiler chickens of II, III, 85

IV and V groups had small taste differences by 0.05; 0.05; 0.11 and 0.12 points were higher than the control group. The average estimate of meat broth of the control group was 4.43 points, in the experimental groups – 4.50; 4.49; 4.56 and 4.56 points. In general, the quantitative and qualitative definition of meat production makes it possible to judge the strength of the effect of feed additives based on vermiculite on the studied indicators, as well as the advisability of their use in feeding the poultry.

Table 24 - Organoleptic evaluation of meat of experimental broiler chickens

Groups Indicators control experimental I II III IV V

Boiled pectoral 4,52±0,14 4,57±0,13 4,53±0,14 4,68±0,11 4,71±0,21 muscles Aroma 4,58±0,14 4,63±0,15 4,62±0,11 4,65±0,16 4,65±0,32 Taste 4,45±0,15 4,47±0,14 4,50±0,14 4,55±0,10 4,57±0,01 Tenderness 4,43±0,11 4,55±0,14 4,53±0,11 4,57±0,14 4,57±0,14 Juiciness 4,50±0,05 4,55±0,06 4,55±0,07 4,61±0,03 4,62±0,05

Average quality assessment Fried pectoral 4,50±0,15 4,53±0,17 4,52±0,12 4,65±0,14 4,65±0,11 muscles Aroma 4,55±0,12 4,55±0,15 4,58±0,13 4,67±0,14 4,69±0,02 Taste 4,60±0,10 4,68±0,11 4,65±0,10 4,70±0,11 4,70±0,17 Tenderness 4,65±0,12 4,67±0,13 4,68±0,12 4,68±0,12 4,69±0,21 Juiciness 4,58±0,09 4,61±0,09 4,61±0,04 4,68±0,06 4,68±0,51

Average quality assessment Broth made from 4,37±0,11 4,48±0,10 4,45±0,13 4,55±0,13 4,55±0,31 pectoral muscles Transparency 4,50±0,14 4,52±0,10 4,53±0,11 4,57±0,11 4,58±0,03 Fortress 4,43±0,10 4,50±0,05 4,49±0,08 4,56±0,12 4,56±0,41

The meat obtained from the experimental birds meets veterinary and sanitary requirements for a quality product and can be sold without restrictions (Appendixe K). Organoleptic assessment of the quality of eggs. Organoleptic tests of eggs were carried out to assess the quality of eggs. Organoleptic evaluation of the product is a generalized result of the evaluation of its quality, performed with the help of the human senses. An organoleptic evaluation can give an opinion about such parameters as the freshness of raw materials; the disruption of the production process is much faster than the instrumental methods.

Organoleptic studies were conducted to study the freshness of eggs. Were evaluated the appearance, the color of the contents of eggs, the smell, taste, consistency and condition of the shell. Table 25 shows the results of organoleptic studies of eggs of experimental groups of hens in comparison with the control group of hens. A total of 300 eggs were examined, obtained three times during the test: at the beginning of the experiment, after 30 days of experience and after 60 days of feeding chickens with feed additives based on vermiculite. Eggs took 20 pieces from each group. In appearance and color of the contents, the eggs of all chickens met the requirements of sanitary quality, all of them were uniform, from white to light yellow, depending on the chickens. The contents of eggs in appearance and color were uniform, light yellow. The smell and taste were also specific, pleasant without foreign odors. All the groups of eggs did not observe a specific taste. The consistency of the contents of eggs is liquid, jelly, without lumps and impurities of blood, stains and other foreign substances were also not observed. The shell of all eggs was round in shape, from white to yellowish-white in color. The shell was clean, free of contamination and mechanical damage. Fresh eggs in the case of light by ovoscope inspection were as follows: the shell was uniform, the air chamber was small and located at the blunt end of the egg. Yolk in the center, its borders are indistinct. While turning the egg, the yolk turned with a slowing down. Inside the egg, no foreign inclusions were found. Ovoscoping of eggs to determine their freshness and exclusion of egg defects was carried out using a special device - an ovoscope. Figure 14 shows the process of ovoscoping of the eggs.

Figure 14 - Ovoscoping of the eggs

Table 25 - Results of organoleptic examination of eggs

Indicators Groups Color Smell Taste Consistency Shell Specific, Liquid, without White-yellowish shade, Specific, lumps and round in shape, without 1 Light yellow color without foreign toothsome admixtures of mechanical damages and odors blood impurities Specific, Jelly like, Yellowish-white in Specific, without lumps color,round in shape, 2 Light yellow color without foreign toothsome and admixtures without mechanical odors of blood damages and impurities Jelly like, Specific, White, round in shape, Specific, without lumps 3 Light yellow color without foreign without mechanical toothsome and admixtures damages and impurities odors of blood Liquid, without Specific, Белые яйца, round in Specific, lumps and 4 Yellow color without foreign shape, without mechanical toothsome admixtures of damages and impurities odors blood Liquid, without Specific, Yellowish-white, round in Specific, lumps and 5 Yellow color without foreign shape, without mechanical toothsome admixtures of damages and impurities odors blood

Thus, according to the results of the veterinary and sanitary examination of the eggs of experimental and control groups of chickens selected according to GOST, the following conclusions can be drawn: - In case of organoleptic examination of samples of eggs of all groups, according to all parameters, they meet the requirements of GOST; - When ovoscoping of the eggs 100% of the fresh eggs of chickens of control and experimental groups of hens were observed, which were fed with feed additives based on vermiculite with the basal diet. From the data obtained in the course of the research, it can be concluded that when the eggs are examined visually, the eggshell is clean, whole, strong. When the ovoscoping, the air chamber was fixed, its height along the major axis was 1,5 mm; yolk occupied a fixed central position, hardly noticeable, contours were not visible; the protein is dense, evenly occupying the entire periphery of the egg, which is translucent. On the basis of the data obtained, it was established that the eggs of all groups of laying hens correspond to the "Norms of Hygienic Requirements for the Quality and Safety of Production Raw Materials and Foodstuffs", according to the principle of sorting, the quality and weight of the egg are of the first category.

Table 26 – Physical and chemical parameters of eggs

Indicators Groups Acid number, mg рН рН КОН/g of white of yolk 1 5,21±0,02 8,92±0,09 5,81±0,02 2 4,92±0,13 8,94±0,53 5,81±0,07 3 4,86±1,24 8,89±0,03 5,83±0,04 4 5,03 ±0,32 8,87±1,23 5,93±1,41 5 4,96±0,05 8,89±1,14 5,98±2,31

Table 26 shows the results of a study of the physical and chemical parameters of laying hens` eggs in a comparative aspect, including the maintenance of the acid number of the yolk, the pH of the yolk and the egg white. The acid number of yolk in the norm is not more than 5-6 (mgKOH)/g. With prolonged feeding of feed containing toxic substances - fat oxidation products (lipid peroxides) that accumulate in the conditions of prolonged or improper storage of mixed fodders and their components, especially fishmeal, meat-bone meal and others, the acid number of yolk rises, which serves as a test for determination of toxic dystrophy of the bird, which leads to a decrease in egg production, a decrease in the biological quality of hatching eggs, a decrease in hatchability and death of embryos during the first days of incubation (30-45%). Young chickens, derived from eggs with high acidity of the

89 yolk and with a minimum content of carotenoids, is born weak, inactive, with difficulty moves and dies within the first 10 days (up to 80%) [201]. Acid number of the yolk did not exceed the maximum permissible standards and met the requirements of state standards. The acid number is lowered especially, where vermiculite was used 3% and 5%, this is due to the fact that vermiculite favorably affects the safety of feed and feed additives, and decrease the acid number. The protein loses water by evaporating it through the pores of the shell, due to the active destruction of ovomycin the stratification of the protein is disturbed and it acquires a liquid consistency. Decrease: height and protein index, number of units Hau, density of eggs; the refraction coefficient rises, partial denaturation of proteins; pH is shifted to the alkaline side to 9.0-9.5, which is accompanied by an almost complete loss of lysozyme activity. Yolk during storage increases in size, it becomes more liquid, its index and refractive index decrease. The membrane of yolk loses elasticity and the pH gradually increases (to 6.8), the decomposition of fats and the decomposition of nitrogen compounds occured. When storing the color of the yolk often changes, it becomes darker, sometimes spotty. In eggs stored for more than 7 days, the acid number of the yolk exceeds the norm (5 mg KOH per 1 g of yolk), the content of vitamin E in the yolk decreases 1.5-2 times. During the study of the pH of the protein and yolk of eggs, it was found that the pH of the protein corresponds to the sanitary norms and rules for storing eggs. Its content does not exceed the maximum permissible standards for fresh chicken`s egg. Depending on the type of groups, the pH of the protein was from 8.87 to 8.94. The pH of the yolk was, respectively, 5.81-5.98. Consequently, the results of the pH of the yolk corresponded to the norm, that is, the eggs of all groups of chickens were benign and fresh.

Table 27 – The results of bacteriological studies of the eggs of control and experimental groups

Group Indicators QMAFAnM/ BG/ cm3 Salmonella/ cm3 Proteus/ cm3 CFU /cm3 1 1,1*105 N.d. N.d. N.d. 2 1,0*105 N.d. N.d. N.d. 3 1,0*105 N.d. N.d. N.d. 4 1,0*105 N.d. N.d. N.d. 5 1,1*105 N.d. N.d. N.d. Note: N.d. - Not detected

To determine the freshness of eggs of control and experimental groups of hens, were studied the microbiological indices of eggs. Microbiological indicators were conducted in accordance with GOST 32149-2013 «Food products of commercial poultry eggs processing. Microbiological analysis methods". Table 27 shows the results of bacteriological examination of the eggs of laying hens of control and 90 experimental groups, which together with the feed received vermiculite and vermiculite with fish meal. According to the results of the QMAFAnM values in eggs of laying hens of all groups vary from 1.0*105 to 1.1*105 CFU/cm3 which indicates a low contamination of eggs and this ratio of bacteria is allowed in eggs. A large number of QMAFAnM is most often indicative of violations of sanitary rules and the technological mode of production, as well as the timing and temperature regimes of storage, transportation and sale of eggs. The bacteria Salmonella, Proteus and Escherichia coli were not found in all samples. The results of QMAFAnM were minor in the bacteriological study of eggs of all groups. Other types of bacteria in egg samples were not detected.

3.7.4 Nutritional value and chemical composition of the meat and eggs of experimental birds Chemical composition of the meat. Determination of the chemical composition and nutritional value of broiler chicken` meat is one of the important tasks of the veterinary-sanitary examination. The chemical composition of broiler chicken meat, which determines its nutritional value and taste, is characterized above all water content, nitrogenous substances, lipids, minerals, carbohydrates and vitamins. The chemical composition of the meat is not constant and essentially depends on the species, physiological state, age, sex, and habitat, the kind of feed and rearing and other factors [202]. The moisture, protein, fat and minerals influence on the nutritional value of broilers and their physiological role as a source of biologically active substances. Supplementation with different feed additives in broiler diet not only improves the aesthetic appearance of the poultry products, but also increases periods of storage and the content of vitamins, minerals, nutrients. Table 28 shows the results of concentration of protein, lipids and ash in broiler meat pectoral muscles of control and experimental groups. Evaluating the quality of broiler meat in this experiments it was found that the protein content in the meat of broilers fed with vermiculite plus fishmeal was higher compared with control group about 5% (group E). The amount of protein in the meat of the experimental groups significantly increased by 0,5% in all group B, C, D and by 5% in group E. Barteczko and Lasek [203] reported that broiler chicken fed with mixtures of higher protein content (23%) showed higher body weight and protein percent in muscle tissue compared to broilers fed with diet (20 and 19%) protein content. The lipids percent in breast fillet without skin were observed to be 0.45% for control group A and for four different experimental groups: 3.0% B; 2.9% C; 4.1% D and 4.3% E, respectively and increased by 0.01%, -0.1%, 1.1% and 1.3%, respectively compared to the control group. Table 28 shows the water content of breast fillet chicken in control and four different groups. The moisture content in the experimental group of birds receiving the feed additives vermiculite and fishmeal was normal. The higher moisture content was observed in broiler chickens fed with 5%V + FM (75.4%), where the highest

91 water content possessed control group. The moisture content was significantly lower in all experimental groups than the moisture content of control breast fillets. A slight increase in moisture content of the meat of the group D and E was observed, which had 0.6% and 1.3% lower moisture than C experimental group (75.4) fed with 5% vermiculite. Researches carried out on the moisture content indicated that vermiculite feed additive influenced on level of meat moisture.

Table 28 - Chemical composition of broiler`s meat (breast) control group (A) and experimental (B-E)

Groups Mean A B C D E Std P value (contr.) (3%V) (5%V) (3%V+FM) (5%V+FM) Error Moisture 75,5 75,1 75,4 74,8 74,1 0,18 0,502 Ash 0,9 0,8 0,9 0,9 0,9 0,02 0,123 Fat 3,0b 3,0a 2,9a 4,1b 4,3b 0,04 0,012 Protein 21,2a 21,0b 21,1b 24,2b 25,0b 0,13 0,033 a-b Means in the same raw with different superscripts are significantly different at P<0,05

The results of these studies indicated a high content of protein in the meat of birds fed with additive of vermiculite with fishmeal that allowed us to conclude that high nutritional value gives a perspective of the use of this feed additive in the production of high quality poultry products (Appendixe L). In the present study, the difference in the protein content of muscle tissue in various experimental groups depended on diets formulated with different levels of protein content and vermiculite. The results of present investigation showed that lipid contribution in breast muscle depended on level of vermiculite and fishmeal in the diet, which agreed with the observations of Osek and Janocha [204]. The research findings of Hanczakowski et al [205] demonstrated that the crude protein and its amino acid content, as well as unsaturated fatty acid profile in fat could influence a cholesterol balance. The ash content in the experimental groups was practically at the same level (in the control group was 1.43% in the first test group was 1.17%). This study confirmed that chemical composition of breast meat depended on type of the diet [206]. Thus, the chemical composition and nutritional value study of meat of broiler chickens in the feed, fed with feed additives based on vermiculite, found that this feed additive was completely harmless, had no negative effects on the chemical composition of broiler meat in the experimental groups. In some experimental groups significantly improved some indicators: protein in the group D and E. The chemical composition assessment of broiler meats, whose ratio included feed additives based on vermiculite of Kazakhstan origin, showed no significant changes. A slight moisture reduction and rise of protein amount has been noted.Thus, the chemical composition and nutritional value study of meat of broilers in the feed, 92 fed with feed additives based on vermiculite, found that this feed additive was completely harmless, had no negative effects on the chemical composition of broiler meat in the experimental groups. In some experimental groups significantly improved some indicators: protein in the group D and E. Chemical composition of the eggs. Determination of the chemical composition and nutritional value of eggs is one of the important components of the veterinary- sanitary examination. The chemical composition influenced on the nutritional value of eggs and described their physiological role as a source of biologically active substances [207 - 212]. The appropriate manipulation with broiler chicken diet could modify fatty acid profile in poultry products and increase its nutritional value. The chemical composition of eggs is characterized above all water content, nitrogenous substances, lipids, minerals, carbohydrates and vitamins and is not constant. Essentially depends on the species, habitat and the kind of feed, rearing and other factors. Table 29 shows the results of concentration of protein, carbohydrates, fat and ash, separately in yolk and white of eggs of control and experimental groups.

Table 29 - Composition and nutritional value of eggs of lying hens fed different levels of vermiculite.

Indicators,% Groups En,val, Dry Carbohyd Moisture Protein Lipids kcal/ matter rates 100g yolk 54,84±0,5 45,27±0,5 15,21±0,3 31,15±0,5 0,66±0,2 344,0 A white 87,13±0,3 12,90±0,5 9,94±0,4 0,26±0,2 0,55±0,2 43,9 yolk 54,53±0,4 45,58±0,5 14,86±0,4 30,38±0,5 0,54±0,1 334,0 B white 87,17±0,3 12,97±0,5 10,14±0,2 0,27±0,1 0,4±0,1 44,2 yolk 53,95±0,5 46,15±0,5 14,95±0,6 29,88±0,5 0,52±0,1 330,0 C white 86,56±0,7 13,53±0,3 9,95±0,7 0,13±0,1 0,54±0,2 43,1 yolk 54,13±0,7 45,98±0,3 16,82±0,4 34,55±0,2 0,7±0,2 381,0 D white 86,35±0,6 13,78±0,8 11,25±0,5 0,36±0,1 0,77±0,1 50,9 yolk 54,37±0,4 45,76±0,4 17,28±0,6 35,87±0,3 0,78±0,2 394,0 E white 86,13±0,3 13,95±4,3 12,13±0,6 0,38±0,1 0,89±0,1 54,9

Evaluating the quality of eggs in this experiments it was found that the protein content in yolk and white of eggs lays fed with V+FM was higher compared with control group of about 2-3% (group D and E). The amount of protein in eggs of the experimental groups showed significant increase by 0.5-1.0 % in group B, C and D and the highest differences were observed between control and E group (2-3%, yolk and white). The lipid percent in yolk were observed to be 31,1% for control group and for: 30.3% B; 29.8% C; 34.5 D and 35.8% E, respectively and increased by 3.4% and 4.7% in group D and E, respectively compared to the control group. The moisture content was significantly lower in all experimental groups than the moisture content 93 of control yolk and white. Contrary, the carbohydrates in yolk and white of hens group D and E were the highest. The dry matter in the experimental groups of yolk was practically at the same level (in the control group was 45.2% in the first test group was 45.5%).

Table 30 - Effect of dietary vermiculite and fishmeal supplementation on the fatty acid profile

D E A B C Fatty acid (FA) (3% (5% (Control) (3% V) (5% V) V+FM) V+FM) myristic C14:0 0,68a 0,61b 0,80a 0,75c 0,57a pentadecanoic C15:0 0,07c 0,08c 0,10a 0,08c 0,19b palmitic C16:0 27,89ab 25,23a 27,39b 27,04a 20,72c margaric C17:0 0,14a 0,16a 0,19c 0,16a 0,43c stearic C18:0 8,04b 7,87ab 6,91d 7,06b 11,16b arachidonic C20:0 0,10b 0,11b 0,14b 0,18b 0,44a docosanoic C22:0 0,15bc 0,19c 0,22b 0,15ab 0,76c Total 37,08a 34,26a 35,74b 35,42a 34,27c SFA myristoleic C14:1 0,17a 0,16b 0,21a 0,15d 0,03a palmitoleic C16:1 6,04b 6,35c 7,16c 5,75d 1,59b oleic C18:1 c-9 37,11a 38,28a 32,97b 37,49a 25,42c C18:1 c- oleic 1,96cd 1,90b 1,95d 2,04b 2,08a 11 eicosenoic C20:1 n-9 0,29b 0,36c 0,26a 0,21ab 0,35b Total b a ab b b 45,57 47,05 42,53 45,65 29,47 MUFA linoleic C18:2 n-6 15,38a 16,47a 18,49b 16,89b 30,92d γ-linolenic C18:3 n-6 0,25c 0,23b 0,31a 0,27c 0,33ab β-linolenic C18:3 n-3 0,57b 0,54c 0,74c 0,67c 2,76a eicosadienoic C20:2 n-6 0,12ab 0,12d 0,22d 0,13d 0,63b arachidonic C20:4 n-6 1,03b 1,32d 1,97a 0,98a 1,62a Total 17,35b 18,69a 21,73b 18,94a 36,26ab PUFA Total n6 17,35a 18,69c 21,73b 18,94ab 36,26b Total n3 0,57a 0,54c 0,74c 0,67a 2,76a PUFA/SF 0,47d 0,55bc 0,61d 0,53a 1,06a A

Thus, the study of chemical composition and nutritional value of hens’ eggs supplementation with mineral and fish meal additives in diet has showed the positive influence on some eggs quality indicators. A higher content of protein, carbohydrates

94 and energy value of eggs were observed. That allowed us to conclude that high nutritional value gives a perspective of the use of this feed additive in the production of high quality poultry products.

3.7.5 Analysis of the fatty acid composition of meat and eggs when used feed additives based on vermiculite Fatty acid composition of meat. The fatty acid composition of chicken lipids depends on different kinds of feed and additives. Many authors have studied how the inclusion of different fat sources in the broiler chickens diet affects the concentration of fatty acid, mainly PUFA in meat. Swierczewska et al. [213212] assume that the quality of meat and mainly fatty acid profile chicken muscles mostly depends on components contained in fed mixtures. Chemical composition of breast meat depended on type of the diet and Castellini et al. [214] found that the organic chickens had carcasses with a higher breast and drumstick percentages and lower abdominal fat levels. However, there are no reports on the effect of levels FA and their type in chicken feeding vermiculite with fishmeal. Feeding with vermiculite and fishmeal led to an increase in total fatty acids (FA) content, decreasing level of saturated fatty acids (SFA) and increasing dietary polyunsaturated fatty acids (PUFA) (Table 30). In the present study the meat of control group contained higher percent of SFA, lower PUFA than experimental groups. The dominant acid was palimitic, oleic, linoleic acids from FA. SFA were ascending and descending. Palmitic and myristic acid decreased about 7% and 0.11% while margaric, stearic, arachidic, docosanoic increased about 0.3%, 3.12%, 0.34% and 0.61%, respectively in group E. The diagramm of SFA, MUFA and PUFA are presented in Figures 15, 16 and 17 (Appendixe M). .

percent totalof fatty acids 10

0 myristic pentadecanoic palmitic margaric stearic arachidic docosanoic A 0,68 0,07 27,89 0,14 8,04 0,1 0,15 B 0,61 0,08 25,23 0,16 7,87 0,11 0,19 C 0,8 0,1 27,39 0,19 6,91 0,14 0,22 D 0,75 0,08 27,04 0,16 7,06 0,18 0,15 E 0,57 0,19 20,72 0,43 11,16 0,44 0,76

Figure 15 - The content of saturated fatty acids in the meat of broiler chickens

percent totalof fatty acids 15

0 myristoleic palmitoleic oleic n-9 oleic n-11 eicosenoic A 0,17 6,04 37,11 1,96 0,29 B 0,16 6,35 38,28 1,9 0,36 C 0,21 7,16 32,97 1,95 0,26 D 0,15 5,75 37,49 2,04 0,21 E 0,03 1,59 25,42 2,08 0,35

Figure 16 - The content of monounsaturated fatty acids in the meat of broiler chickens

15 percent totalof fatty acids 10

0 linoleic γ-linolenic β-linolenic eicosadienoic arachidonic A 15,38 0,25 0,57 0,12 1,03 B 16,47 0,23 0,54 0,12 0,32 C 18,49 0,31 0,74 0,22 1,97 D 16,89 0,27 0,67 0,13 0,98 E 30,92 0,33 2,76 0,63 1,62

Figure 17 - The content of polyunsaturated fatty acids in the meat of broiler chickens 98

General tendency of similarity in percent of FA in groups B and D fed with 3% vermiculite with or without fishmeal addition contrary to groups C and E indicating differences in FA content. Linoleic acid from PUFA significantly increased over 50% in group E than other groups, α-linolenic 5 times higher in group E than in control, which represented a great contribution to the sum of PUFA. The SFA synthesis is inhibited in the liver more during digestion of unsaturated fats than saturated fats. Also, the increase of PUFA decreased the synthesis of monosaturated MUFA by inhibiting the activity of 9-desaturase complex which is the key enzyme needed to convert SFA to MFA. The results of current study indicated that the FA profile broiler tissue was customized by a diet a mineral and fishmeal. Feeding with different kinds of diet containing 3% and 5% ratios of vermiculite and fishmeal can be possible to apply in future, which was proven by these results including the chickens tissue level of specific fatty acid or mixture of fatty acids thought to be beneficial to human health (e.g. oleic acid). The fatty acid composition of chicken lipids depends on different kinds of feed and additives. Many authors have studied how the inclusion of different fat sources in the broilers diet affects the concentration of fatty acid, mainly PUFA in meat [220]. However, there are no reports on the effect of levels FA and their type in chicken feeding vermiculite with fishmeal. This research provides the first comprehensive demonstration of vermiculite from Kazakhstan as feed additive on broilers and hens performance and meat quality. Vermiculite was not investigated so far as feed additive and had no toxicity and beneficially influenced on fishmeal. For future poultry industry, especially in developing countries, this novel knowledge of vermiculite as feed additive is very important for better understanding how to improve meat properties and quality ensuring consumers' health and safety. Fatty acid composition of egg`s yolk. Many authors have studied the effect of different fat sources in the broilers diet on type of concentration of fatty acid, mainly PUFA in the meat [216, 217]. However, there are no reports of the effect of levels FA in eggs mineral diet of hens and this study showed the influence of vermiculite supplementation on the fatty acid level of egg`s yolk. The hens have the ability to deposit dietary lipids into the egg yolk and to modify the fatty acid (FA) composition of the egg [218]Ошибка! Источник ссылки не найден.. Dietary mineral plus protein sources had a significant effect on the FA profile in yolk (Table 31). Feeding with supplementing diet led to an increase in total FA content, a little changing of level of saturated fatty acids (SFA), increasing dietary mono (MUFA) and polyunsaturated fatty acids (PUFA). Regardless of V+FM inclusion levels or combination, contributed to a significant increase in the concentrations of C18:2 n-6 and C18:3 n-3 fatty acids and total PUFAs in yolk lipids. The diagramm of SFA, MUFA and PUFA of yolk are presented in Figures 18, 19 and 20.

Table 31 - Fatty acid composition of egg`s yolk (control and experimental groups, g 100 g-1)

Fatty acid Groups composition, A B C D E g 100 g-1

C14:0 0,380,07 0,540,06 0,220,07 0,220,04 0,270,07 C15:0 0,080,02 0,070,01 0,070,02 0,090,01 0,080,02 C15:0 iso n.d n.d n.d 0,010,00 0,010,00

C15:0 aiso n.d n.d n.d 0,010,00 0,010,00

C16:0 iso n.d n.d n.d 0,030,01 0,010,03 C16:0 27,230,87 23,420,72 23,031,02 26.190.97 26.971.64

C17:0 0,290,04 0,280,05 0,280,03 0.300.09 0.340.05

C18:0 10,860,34 10,20,28 9,490,14 11,860,64 11,060,12 C20:0 0,060,01 0,060,01 0,060,02 0,040,01 0,040,01

C22:0 0,050,01 0,030,01 0,040,01 0,100,02 0,100,03 C14:1n-5 0,02 0,00 0,010,00 0,010,02 0,090,02 0,040,01 C16:1 n-7 0,060,01 0,060,02 0,050,01 0,09  0,01 0,040,01

C17:1 0,110,03 0,110,02 0,100,02 0,140,03 0,160,04 C t 18:1 0,110,02 0,070,01 0,070,01 0,440,09 0,250,03 6,9,10,11,12

C18:1 c9 25,870,75 28,390,68 26,240,28 41,371,24 42,462,61 C18:1 c11 1,890,23 1,890,15 1,900,11 2,380,21 2,390,21 C18:1 c13 0,140,07 0,120,04 0,110,03 0,130,05 0,140,04 C18:1 t 16 n.d n.d n.d 0,090,02 0,080,02

C20:1 n-9 0,210,06 0,180,03 0,200,02 0,130,02 0,340,07

C18:2 t13c9 0,010,00 0,010,00 0,010,02 0,060,01 0,020,01

C18:2 t12c9 0,040,02 0,030,01 0,030,01 0,050,01 0,050,01 C18:2 t11c15 0,010,01 0,010,00 n.d 0,030,01 0,020,01

C18:2 n-6 8,650,65 8,760,36 25,690,79 28,401,78 31,551,54

C18:3 n-3 0,370,09 0,330,04 1,820,48 1,980,34 2,410,24 C18:2 c9t11 n.d n.d n.d 0,070,01 0,070,01

C20:2 n-6 0,110,03 0,330,07 0,100,02 0,330,06 0,460,07 C20:4 n-6 1,920,12 1,850,14 1,830,03 2,140,05 3,510,11

C22:5 n-3 0,280,08 0,150,04 0,150,02 0,610,05 0,930,9

C22:6 n-3 1,050,11 1,110,09 1,070,07 1,150,07 1,510,08 Total SFA 38,95 34,61 33,19 38,85 38,89 Total MUFA 28,41 30,83 28,68 44,86 45,90 Total PUFA 12,44 12,58 30,70 34,82 40,53 n.d – not detected

100

30 25 behenic 20 arachidic stearic 15 margaric palmitic fatty acids fatty 10 palmitic iso percent of total percenttotal of pentadecanoic aiso 5 pentadecanoic iso pentadecanoic 0 myristic A B C D E myristic 0,38 0,54 0,22 0,22 0,27 pentadecanoic 0,08 0,07 0,07 0,09 0,08 pentadecanoic iso 0 0 0 0,01 0,01 pentadecanoic aiso 0 0 0 0,01 0,01 palmitic iso 0 0 0 0,03 0,01 palmitic 27,23 23,42 23,03 26,19 26,97 margaric 0,29 0,28 0,28 0,3 0,34 stearic 10,86 10,2 9,49 11,86 11,06 arachidic 0,06 0,06 0,06 0,04 0,04 behenic 0,05 0,03 0,04 0,1 0,1

Figure18 - The content of saturated fatty acids in the egg`s yolk

101

45 40 35

100г 30 \ eicosenic n-9 25 oleic t-16 20 oleic c-13 oleic c-11 15 oleic c-9 10 oleic t 6,9,10,11,12 margaric 5 palmitoleic 0 miristoleic A B C D E miristoleic 0,02 0,01 0,01 0,09 0,04

palmitoleicколичествожирных кислот, г 0,06 0,06 0,05 0,09 0,04 margaric 0,11 0,11 0,1 0,14 0,16 oleic t 6,9,10,11,12 0,11 0,07 0,07 0,44 0,25 oleic c-9 25,87 28,39 26,24 41,37 42,46 oleic c-11 1,89 1,89 1,9 2,38 2,39 oleic c-13 0,14 0,12 0,11 0,13 0,14 oleic t-16 0 0 0 0,09 0,08 eicosenic n-9 0,21 0,18 0,2 0,13 0,34

Figure 19 - The content of monounsaturated fatty acids in the egg`s yolk

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35 30 25 docosahexaenoic n-3 docosadienoic n-3 20 arachidonic n-6 eicosadienoic n-6 15 linoleic c9t11 3 α-linolenic acid 10 linoleic n-6 linoleic t11c15 5 linoleic t12c9 0 linoleic t13c9 количествожирных кислот, г/100г A B C D E linoleic t13c9 0,01 0,01 0,01 0,06 0,02 linoleic t12c9 0,04 0,03 0,03 0,05 0,05 linoleic t11c15 0,01 0,01 0 0,03 0,02 linoleic n-6 8,65 8,76 25,69 28,4 31,55 3 α-linolenic acid 0,37 0,33 1,82 1,98 2,41 linoleic c9t11 0 0 0 0,07 0,07 eicosadienoic n-6 0,11 0,33 0,1 0,33 0,46 arachidonic n-6 1,92 1,85 1,83 2,14 3,51 docosadienoic n-3 0,28 0,15 0,15 0,61 0,93 docosahexaenoic n-3 1,05 1,11 1,07 1,15 1,51

Figure 20 - The content of polyunsaturated fatty acids in the egg`s yolk

103

The SFA: palmitic, arachidic acid decreased about 0.1-0.3% while margaric, stearic, docosanoic increased about 0.2×0.5% in group E. Addition of vermiculite increased clearly the level of myristic (C14:0) acid in yolk eggsin group B (0.54%). A slight increase of stearic acid was observed when 5% V was added. No significant difference in palmitic acid content was observed compared with control. When 3% and 5% of V+FM were added, the percentage of 18:1 oleic acid increased (near two times in group D and E). There is no significant increase of 16:1 palmitoleic acid percentage. Similar results were reported by [217]. Our data shows that the addition of a natural mineral with fish meal significantly increased in yolk eggs the level of total n-3 fatty acids (Table 31), with this response being primarily related to higher levels of linolenic acid. Similar results were also reported by [217] using fed diets containing microencapsulated fish oil. Fish oil is one of the best known sources of n- 3 PUFA, as it is rich in EPA and DHA. Especially, it is important to note that PUFAs (linoleic acid and alpha-linolenic acid) have displayed protection against lipid per oxidation increasing the levels of several cellular antioxidants such as ascorbic acid, α-tocopherol. The linoleic acid significantly increased over tree and four times ore in group C, D and E than in control and B group, and gamma-linolenic seven times higher in group E, which represented a great contribution to the sum of PUFA. Most n-6 polygene fatty acids were noted in the yolk eggs hens fed with the V+FM feed. However, this content was higher in all the experimental groups in comparison to the control group. The results of current study indicated that the FA profile in yolk eggs is customized by a diet with mineral and fishmeal. Feeding with different kinds of diets containing 3% and 5% ratios of vermiculite and fishmeal can be possible to apply in future which was proven by these results including the egg’s yolk level of specific fatty acid or mixture of fatty acids thought to be beneficial to human health (e.g. oleic acid). The study of the chemical composition and nutritional value of eggs from hens’ supplementation with mineral and fishmeal additives in diet has showed the positive influence on eggs quality indicators and morphometric parameters. A higher content of protein, carbohydrates and energy value of eggs were observed. The supplementing diet had a significant effect on the fatty acid profile of yolk amino acids. Feeding with supplementing diet in mineral and protein led to an increase in total fatty acids content and dietary mono (MUFA) and polyunsaturated fatty acids (PUFA). The vermiculite contributed to a significant increase in the concentrations of C18:2 n-6 and C18:3 n-3 fatty acids and total PUFAs in yolk lipids.

3.7.6 Aminoacid composition of meat and eggs The content of nonessential and essential amino acids in the meat when using feed additives based on vermiculite. Poultry meat contains all the necessary substances for human nutrition, it is the source of the basic nutrients (proteins, animal fats, mineral and extractive substances), which are presented in the most optimal quantitative ratio and easily absorbed by the human body. And the greatest value for

104 consumers of poultry meat is proteins, consisting of interchangeable and essential amino acids. The number of different essential and non-essential amino acids in meat proteins determines its nutritional value and biological value. The content of essential amino acids in proteins of poultry meat depends on the content of amino acids, primarily in feed, because the organism of agricultural birds is not able to synthesize them. The amino acid composition of the protein of poultry meat can be changed by including various biological additives in the main diet or by using non-traditional feeds. Therefore, the aim of our work was to study the chemical composition and biological value of meat of broiler chickens who received feed additives based on vermiculite in order to increase their fattening intensity. When studying the amino acid composition of the pectoral muscles of white meat of broiler chickens of the experimental and control groups, we determined the content of 19 amino acids, 8 of which are indispensable (Appendixe N). . These data are presented in table 32. From the table, it can be seen that in the white meat of broilers of the experimental groups D and E, in comparison with the white meat of the control chicks, the content of individual essential amino acids increased by 0,56-0,79 mg/100g, or on average by 8.0-10.9 %, including arginine - by 0.1-0.14 g/100g (8.0-10.8%), isoleucine – 0.20-0.21g/100g (21.0-21.8%), threonine – 0.03-0.11g/100g (3.7-12.3%), tryptophan – 0.16-0.22g/100g (30.7-37.9%). In the experimental groups B and C, the valine content decreased by 0.03-0.02g/100g (3.5- 2.3%), lysine by 0.02-0.01g/100 g (1.4-0.7%), methionine by 0.03-0.04g/100g (6.6- 8.9%), phenylalanine by 0.02g/100g (3.0%). However, on average, the amount of essential amino acids increased only by 0.31g/100g or by 4.6%. In groups D and E of nonessential amino acids, the increase was noted in the histidine content by 0.18-0.13g/100g (18,7-14,3%), leucine 0.18-0.27g/100g (10.6- 15.2%) and cystine by 0.018-0.029g/100g or by 43.9-55.7%. The content of glycine in test group B was reduced by 0.03mg/100g, or 1.96% compared to the control group. At the same time, the amount of nonessential amino acids increased only by 1.004g/100g, or by 8.5%. The ratio of the sum of essential amino acids to the nonessential in the meat of broilers of the experimental group was 1.74, in the control chicken meat -1.67 or 4.02% higher. To assess the biological value of broiler meat by the amino acid composition, the ratio of the amount of tryptophan (the index of the content of muscle proteins) to hydroxyproline (an index of inferior connective tissue proteins) was determined. In the control group, this ratio was 5.2, and in the experimental groups: B-5.3; C-5.3; D-5.5; E-5.9 or 15,4% higher than the control group, which indicates a significant improvement in food and consumer properties of meat. The amino acid composition of broiler chickens` the meat receiving feed additives based on vermiculite was compared with the reference protein values taken for the standard, and the percentage of (score) of each amino acid in the protein was determined. By calculation of amino acid scores, we found that in meat proteins of broilers of experimental groups, there was one limiting amino acid, tryptophan.

105

Table 32 - Amino acid composition of broiler chickens` meat

Amino acid, g/100g Control (n=5) Experimental groups (n=20) A B C D E Essential amino acids Arginine 1,15 ± 0,2 1,14± 0,1 1,16±0,3 1,25±0,3 1,29±0,3 Valine 0,84 ± 0,1 0,81 ± 0,1 0,82±0,2 0,87±0,4 0,86±0,3 Isoleipin 0,75 ± 0,1 0,76± 0,3 0,88±0,2 0,95±0,1 0,96±0,2 Lysine 1,34 ± 0,1 1,32 ± 0,4 1,33±0,2 1,35±0,1 1,38±0,2 Methionine 0,45± 0,2 0,42 ± 0,4 0,41±0,1 0,48±0,5 0,47±0,2 Threonine 0,78 ± 0,4 0,78 ± 0,7 0,81±0,3 0,81±0,4 0,89±0,3 Tryptophan 0,36 ± 0,1 0,39 ± 0,5 0,38±0,7 0,52±0,3 0,58±0,1 Phenylalanine 0,66 ± 0,2 0,64 ± 0,6 0,67±0,4 0,75±0,2 0,78±0,4 Amount of essential amino acids 6,42 6,26 6,46 6,98 7,21 Nonessential amino acids Alanin 0,57 ± 0,1 0,93 ± 0,4 0,95±0,5 1,12±0,5 1,14±0,3 Aspartic acid 1,43 ± 0,1 1,47 ± 0,3 1,48±0,1 1,69±0,3 1,85±0,4 Histidine 0,78 ± 0,2 0,82 ± 0,5 0,75±0,2 0,96±0,2 0,91±0,5 Glycine 1,53 ± 0,1 1,50 ± 0,1 1,52±0,2 1,56±0,2 1,62±0,5 Glutamic acid 2,73 ± 0,2 2,68 ± 0,2 2,75±0,5 2,75±0,3 2,84±0,3 Leucine 1,51 ± 0,2 1,54 ± 0,4 1,50±0,3 1,69±0,3 1,78±0,5 Oxyproline 0,069 ± 0,2 0,073 ± 0,1 0,071±0,1 0,094±0,4 0,098±0,1 Proline 0,75 ± 0,1 0,63 ± 0,1 0,68±0,2 0,78±0,1 0,81±0,3 Serin 0,71 ± 0,4 0,71 ± 0,3 0,73±0,1 0,85±0,1 0,96±0,2 Tyrosine 0,67 ± 0,5 0,65 ± 0,2 0,66±0,3 0,69±0,1 0,68±0,2 Cystine 0,023 ± 0,1 0,025 ± 0,1 0,022±0,3 0,041±0,1 0,052±0,2 Amount of nonessential 10,772 11,028 11,113 12,225 12,740 amino acids

106

In practice, the value of muscle proteins or protein-quality the indicator (PCI) is determined by the ratio of tryptophan to hydroxyproline. Tryptophan is found only in high-grade proteins, and oxyproline is more in connective tissue proteins. It is believed that the greater the ratio of tryptophan to hydroxyproline, the higher the biological value of meat proteins. The ratio of tryptophan to hydroxyproline in the white muscles of broilers can be up to 5-7, and in the red muscles - about 3-8. By the ratio of tryptophan to hydroxyproline, that is full-fledged proteins to inferior, the meat of broiler chickens surpasses the meat of other farm animals. The ratio of tryptophan to hydroxyproline in broilers` meat of experimental groups averaged 5,5; in control chicken meat – 5.2; this is 15.4% higher, which indicates the prospect of using vermiculite in the form of feed additives during industrial cultivation and fattening of broiler chickens. The amino acid in broiler meat proteins when using a feed additive is the amino acid from the nonessential group - tryptophan, which confirms the tendency of increasing the protein-quality index of broiler meat when using vermiculite with fishmeal. The content of nonessential and essential amino acids in the egg when using feed additives based on vermiculite. Amino acid composition is the quantitative content of individual amino acids in the protein. The amino acid composition of the protein is examined to assess the biological value of the product. The amino acid composition of protein depending on the type of product, the species of the poultry and the animal [220]. The purpose of this study was to study the amino acid composition of the eggs of laying hens of control and experimental groups. Table 33 shows the results of studying the amino acid composition of laying hens` eggs. During the study, it was found that in the experimental groups’ egg the content of valine, leucine, lysine, methionine, threonine and phenylalanine is higher than in the amino acid composition of the eggs of control group. All these amino acids are irreplaceable and therefore they are very important for the human body (Appendixe O). Of the non-essential amino acids, the content of alanine in the egg of the control group was 0,87g/100g, and in eggs of the experimental groups D and E its content was significantly higher and amounted to 0.95g/100g. The content of aspartic acid, glutamic acid and cystine of the test group C was lower in comparison with the eggs of the control group. The content of glycine and glutamic amino acids was higher in the eggs of the experimental group B and amounted to 0.55 and 2.08g/100g, respectively, as compared with the control group. The tyrosine content in the eggs of the group D and E was 0.89 – 0.96g/100g, and in the control group 0.71g/100g, which is higher by 0.22g/100g.

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Table 33 - Amino acid composition of eggs` yolk

Amino acid, g/100 g: Groups A B C D E Essential amino acids Valine 0,69±0,09 0,56±0,23 0,70±0,11 0,89±0,21 0,91±0,04 Isoleucine 0,58±0,05 0,58±0,21 0,62±0,32 0,69±0,14 0,70±0,23 Leucine 0,86±0,01 0,84±0,02 0,85±0,02 0,90±0,04 0,98±0,01 Lysine 0,42±0,02 0,45±0,02 0,35±0,04 0,52±0,02 0,55±0,02 Methionine 0,67±0,12 0,70±0,31 0,68±0,04 0,86±0,35 0,87±0,03 Threonine 0,59±0,04 0,59±0,12 0,59±0,05 0,74±0,15 0,76±0,04 Tryptophan 0,19±0,07 0,18±0,41 0,18±0,01 0,36±0,21 0,41±0,01 Phenylalanine 0,63±0,09 0,65±0,03 0,65±0,32 0,75±0,01 0,78±0,32 Total 4,63±0,04 4,55±0,12 4,62±0,32 5,71±0,44 5,96±0,03 Nonessential amino acids Alanin 0,85±0,05 0,87±0,01 0,86±0,32 0,95±0,52 0,95±0,21 Arginine 1,16±0,03 1,17±0,32 1,19±0,41 1,26±0,22 1,25±0,22 Aspartic acid 1,34±0,01 1,32±0,12 1,33±0,53 1,45±0,14 1,49±0,32 Histidine 0,39±0,04 0,39±0,03 0,39±0,54 0,48±0,02 0,47±0,41 Glycine 0,51±0,03 0,55±0,21 0,52±0,14 0,74±0,14 0,72±0,21 Glutamic acid 2,05±0,05 2,08±0,01 2,04±0,12 2,18±0,03 2,21±0,11 Oxyproline 0,38±0,22 0,38±0,01 0,41±0,32 0,47±0,03 0,46±0,02 Proline 0,73±0,05 0,74±0,02 0,73±0,33 0,86±0,21 0,95±0,32 Serin 1,37±0,01 1,38±0,02 1,39±0,21 1,51±0,13 1,48±0,21 Tyrosine 0,71±0,11 0,74±0,51 0,75±0,34 0,89±0,21 0,96±0,01 Cystine 0,28±0,03 0,28±0,53 0,26±0,14 0,31±0,32 0,30±0,32 Total 9,77±0,01 9,91±0,32 9,87±0,41 11,1±0,33 11,24±0,04 Amount of all amino acids 14,4±0,05 14,46±44 14,49±0,73 16,81±0,77 17,2±0,07

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Figure 21 shows the results of a study of the quantitative content of nonessential and essential amino acids of laying hens` eggs. In a comparative evaluation of amino acid parameters, it was found that the total number of amino acids prevails in the eggs of the experimental groups that received vermiculite with fish meal along with the main diet and is equal to 15.74g/100g, and in the hens of the control group the total amino acid content is significantly lower than in eggs of experimental groups and is 14.4g/100g, respectively.

12 11,1 11,24 10 9,77 9,91 9,87

8 A 6 5,71 5,96 4,63 4,55 4,62 B 4 C D 2 E

essential amino acids

non-essential amino acids

Figure 21 - The content of the total number of nonessential and essential amino acids of laying hens` eggs, g/100g

The parameters of the amino acid composition of eggs of the experimental and control groups of hens were insignificant that fodder additives based on vermiculite do not negatively affect the amino acid composition of eggs. And this shows that it is possible to use vermiculite as a fodder additive in the feeding of laying hens.

3.7.7 Vitamin content of the meat and eggs while used feed additives containing vermiculite Vitamin content of the meat. Vitamins are low-molecular organic acids which are formed in plants. They are very important for the life of the human body and animals. Absence of vitamins or its low content in feeds leads to disruption of metabolism, to various diseases (rickets, polyneuritis, night blindness and others). In the studies the content of vitamins in chicken meat, as used feed additives based on vermiculite, it was established that feed additives do not adversely affect the vitamin content in the experimental groups of birds. This is the reason for using these feed additives in broiler feeds. The vitamins in the meat of chicken enter to our body. Many water-soluble and part of fat-soluble 109 vitamins are part of the enzymatic systems. Many vitamins in our body are converted into coenzymes. Coenzyme is a substance that binds to an enzyme for its greater activation. Enzyme complexes accelerate the most diverse chemical reactions in the body. With their help, the metabolism is regulated, certain processes are triggered, some substances are cleaved and others are formed. Table 34 presents the results of analysis vitamin content of broiler meat when used as a component of vermiculite feed. When studying the content of vitamins in the meat of broilers of experimental groups a decrease the level of vitamins was not observed to compare with control group. Depending on the type of vitamins, there was a tendency to increase the level of vitamins, especially when feeding 3% and 5% of vermiculite with fishmeal. If the content of vitamin A in the control group was 0.06 ± 0.03mg/100g, in the experimental group E (5% of vermiculite with fish meal was added) its value was 0.08 ± 0.2mg/100g. The content of vitamin A in the 4 experimental group increased by 0.02 mg / 100 g compared with the control group. In terms of thiamine content, no noticeable octogenations from the norm were observed in the experimental groups of birds, their number was in the range 0.06 ± 0.001mg to 0.07 ± 0.01mg/100g. The content of riboflavin in the control and experimental groups of broilers was also within the normal range, and ranged from 0.15 ± 0.002mg/100g to 0.17 ± 0.08mg/100g. In the study, a noticeable increase in the level of vitamin PP was observed with an increase in the percentage of the feed admixture of vermiculite with fish meal to the feed. The highest level of niacin was observed in the experimental group E which broiler chickens were fed 5% V + FM (Appendixe P).

Table 34 - Vitamin content in the meat of broiler chickens

Vitamins Control group Experimental groups mg/100g A B C D E А 0,06±0,03 0.05±0,31 0.06±0,01 0.07±0,02 0.08±0,2 В1 0,06±0,05 0,06±0,04 0,06±0,001 0,07±0,012 0,07±0,01 В2 0,15±0,002 0,15±0,008 0,16±0,004 0,17±0,01 0,17±0,08 В3/РР 5.6±0,02 5,4±0,008 5,6±0,01 5,8±0,003 5,9±0,03 В5 0,7±0,01 0,7±0,04 0,7±0,006 0,8±0,06 0,8±0,07 В6 0,5±0,01 0,4±0,01 0,6±0,02 0,7±0,08 0,8±0,11 В9 0,003±0,003 0.003±0,01 0.003±0,03 0.005±0,04 0.005±0,02 С 1,9±0,1 1,9±0,3 1,9±0,1 2,0±0,2 2,0±0,1

The level of niacin did not exceed the norm for chickens. The level of pantothenic acid (B5), folic acid (B9) and ascorbic acid (C) in the control and experimental groups of broiler chickens also remained normal. There were no abnormalities in the level of pyridoxine content, there was a noticeable increase in its content in the experimental groups as compared to the control group, its peak reached in the third and fourth experimental group 0.7 ± 0,08mg/100g and 0.8 ± 0,11mg/100g respectively. 110

According to the results of the analysis the content of vitamins of meat, it was established that the vitamin composition of chicken meat was not subjected to pathological changes. All indicators were within norms for a species of birds. We noticed a slight increase in the level of niacin in the experimental groups, which is probably due to the fact that the fish meal that fed the birds is rich in this type of vitamin. The maximum amount of niacin was in the fourth experimental group of broiler chickens, which were fed a feed containing 5% vermiculite with fishmeal. Vitamin content of the eggs. The nutritional and biological value of food eggs is determined by the content of vitamins, which are irreplaceable nutritional factors in the metabolism of the organism. As shown in the literature data in the largest amount of water-soluble vitamins in the egg contains vitamin B2. From the above it follows that the determination of the content of vitamins in eggs is one of the most important tasks (Appendixe Q).

Table 35 - The content of vitamins in the eggs of control (A) and experimental groups (B-E), mg/100g (n = 10)

Vitamins Groups mg/100g A B C D E

A 0,25±0,03 0,25±0,3 0,26±0,01 0,28±0,6 0,28±0,2 B1 0,07±0,6 0,06±0,01 0,07±0,02 0,09±0,2 0,09±0,03 B2 0,45±0,4 0,46±0,2 0,45±0,02 0,47±0,3 0,49±0,1 B3 / PP 3,6±0,04 3,6±0,1 3,6±0,4 3,9±0,4 3,9±0,01 B5 1,4±0,05 1,5±0,01 1,6±0,4 1,6±0,04 1,7±0,2 B6 0,13±0,04 0,12±0,3 0,13±0,3 0,18±0,3 0,18±0,02 E 0,6±0,01 0,6±0,4 0,7±0,2 0,8±0,02 0,8±0,1

According to the literature, the lack of vitamin A disrupts the growth and development of the body, the mucous membrane and skin function is disrupted. Our research shows that (Table 35) the vitamin A is contained in the control group an amount of 0.25 ± 0.03mg/100g, its quantitative content in eggs of experimental chicken groups is: B – 0.25 ± 0.3mg/100g, C – 0.26 ± 0.01 mg/100g, D – 0.28 ± 0.6 mg/100g, E – 0.28 ± 0.2 mg/100g, which is 2.8% higher than in the control group. The amount of vitamin B1 in the control eggs was 0.07 ± 0.6 mg/100g, this value in the experimental group E was 0.09 ± 0.03mg/100g. The content of vitamin B2 in the eggs of the experimental groups averaged 0.47 ± 0.05mg/100g, which in the control group was 0.45 ± 0.4 mg/100g. The vitamin B5 content (pantothenic acid), which regulates the synthesis of hemoglobin in the eggs of the control group was 1,4 ± 0,05mg/100g, and in eggs of experimental hens its value is 1.5 ± 0.01; 1.6 ± 0.4; 1.6 ± 0.04; 1.7 ± 0.2mg/100g respectively. Niacin or vitamin PP is a vitamin that provides stable functioning of the human brain, its nervous system. With its insufficiency, fatigue, weakness and growth retardation occur. Therefore,

111 determining the content of vitamin PP is very important. The quantitative content of vitamin PP in eggs ranged from 3.6 ± 0.04 mg/100g to 3.9 ± 0.4 mg/100g. In conclusion, it should be noted that when determining the content of vitamins in eggs of experimental chicken groups, it is established that the percentage of vitamin E is higher by 17.2%, and vitamin B6 is higher by 14.7% compared to the control group.

3.7.8 Mineral content of meat and eggs Mineral content of the meat. Muscles of birds are rich in microelements, among them is potassium, sulfur, phosphorus, nartium, chlorine, calcium, which is allocated by the quantity, as well as trace elements: iron, zinc, copper, fluorine that play an important role in metabolism [222]. The bulk element is in association with proteins and other constituents of meat, which contributes to their high digestibility. In turn mineral substances activate protein digestibility and assimilability, which distinguishes them from similar ones contained in vegetable products or mineral feeds. The findings of the mineral composition of broiler chicken meat are presented in table 36.

Table 36 - Quantitative content of macro and microelements in meat of broiler chickens

Groups 1 2 3 4 5 indexes К, g/kg 3,32±0,1 3,47±0,1 3,50±0,2 3,49±0,1 3,54±0,4 Ca, % 0,12±0,10 0,21±0,05 0,40±0,03 0,13±0,23 0,14±0,21 Mg, g/kg 0,36±0,05 0,25±0,01 0,41±0,03 0,45±0,01 0,63±0,2 P,% 0,91±0,01 0,82±0,03 0,94±0,04 1,14±0,10 1,31±0,02 Cu, mg/kg 0,46±0,1 0,47±1,6 0,64±0,9 0,44±0,3 0,48±0,5 Na, g/kg 0,88±0,1 0,87±0,4 0,75±0,4 0,94±0,6 0,98±0,1 Fe, mg/kg 12,4±2,1 13,6±1,6 13,9±1,5 12,4±2,2 12,5±3,1 Mn, mg/kg 0,33±0,1 0,33±1,1 0,35±0,5 0,37±0,3 0,39±0,1

By analyzing obtained data of the mineral composition of meat, against the background of general decline in mineral substances in the meat of the experimental groups were not certain trends. There was an increase in the content of calcium by 0.02% and phosphorus by 0.4%, respectively. In the third group the pattern was more pronounced and the difference with the control was 0.40%, and 0.84%, calcium – 0.28%, phosphorus – 0.03%. In the fourth group were obtained results similar to the third. An increase in the content of these two the most important mineral substances contributes to an increase in the biological value of meat, since calcium is involved in the regulation of vascular endothelial vascularity, in the creation of bone tissue structure, and in blood coagulation. It stimulates the activity of the heart muscle, lowers the permeability of cell membranes, and participates in the regulation of the activity of many enzymes. Due to the increase in phosphorus content, the meat of 112 experimental birds becomes more valuable, since phosphorus is an integral part of bones and teeth, a component of nucleic acids, phosphoproteins and phosphotides, is part of buffer systems, macroergic phosphates, participates in many metabolic reactions, primarily glycolysis, glycogenolysis and oxidative phosphorylation. The biological significance of microelements in the animal organism is well known. Microelements actively participate in the basic functions of the body: the processes of growth, development, reproduction and maintenance of health and productivity. The results of the study of the content of iron, manganese and copper in the muscle tissue of chickens of the experimental groups indicate that the amount of iron was higher in the birds of the experimental groups. Thus, in the second experimental groups, this index in red muscle tissue exceeded the control one by 1.2 mg, in the third group - by 1.5 mg, and in the 5-th - by 0.1 mg. The content of iron in white muscle tissue in the second experimental group exceeded the control by 8.8%, while in the 3rd – 10.8%. This is obviously due to the fact that in these chickens under the influence of natural mineral most intensively proceeded processes of hematopoiesis, since iron plays an important role in the hematopoietic processes. According to the content of manganese and copper, the meat of the experimental chickens did not differ, although there was a tendency to increase these values in the experimental groups. Thus, the use vermiculite had a positive effect on the state of mineral metabolism of the organism broiler chickens. A certain increase in the level of intensity of metabolic processes led to changes in the hematological pattern and biochemical indices of muscle tissue, and besides the natural mineral influenced mainly on the metabolism, the protective mechanisms of the poultry organism, and the assimilation of essential nutrients that are eventually affected the quality of poultry meat. After analyzing the obtained indexes of mineral composition of chicken meat in feed supplemented with feed additives based on vermiculite, we came to the conclusion that there was no definite tendency for a general decrease in mineral substances in the meat of broiler chickens of experimental groups. Natural vermiculites in the feeding of broiler chickens are effective in cases when feeds containing vermiculite with a mass fraction of 5% during the growing period are used in broiler feeding. As can be seen from the obtained data, the use of mixed fodders containing vermiculite in feeding chicken broilers is effective. Significant economic effect in this case is manifested by saving feed, reducing morbidity, improving product quality. Mineral content of the eggs. Macroelements are chemical elements, which are calculated in the human body by grams. The macroelements include calcium, phosphorus, magnesium, potassium, chlorine, iron and others [223]. The amount of minerals in the body is measured in micrograms. Macro and microelements provide the work of the main body systems (muscle - participate in the process of muscle contraction, digestive and cardiovascular). Their lack or absence can lead to both serious illnesses and to the death of the body. Therefore, the aim of our studies was to study the macro and microelement composition of eggs of chickens, which fed with basal diet including vermiculite and vermiculite with fish flour in different percentage quantities.

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Table 37 - Indicators of mineral content in eggs, mg/100g (n = 10)

Minerals Groups 1 2 3 4 5 Sodium 130±0,25 134±0,53 138±0,52 135±0,12 138±0,14 Cali 136±0,02 140±0,32 154±0,42 145±0,06 148±0,04 Calcium 55±0,14 58±0,91 61±0,13 55±0,23 56±0,51 Magnesium 13±0,32 12±0,83 14±0,65 14±0,76 15±0,03 Phosphorus 185±0,65 188±0,09 195±0,84 226±0,18 235±0,01 Iron 2,3±0,98 2,5±0,06 3,9±0,91 3,6±0,03 3,9±0,62 Iodine 0,22±0,12 0,22±0,84 0,23±0,05 0,23±0,03 0,23±0,34 Zinc 1,2±0,02 1,1±0,05 1,3±0,21 1,3±0,03 1,4±0,02

Table 37 shows the results of the study of minerals in the eggs of control and experimental groups. In the course of the study it was found that the sodium content in the eggs of the control group was 130 ± 0.25mg, while in the eggs of the experimental group 2 it was only 134 ± 0.53mg, in the third group 138 ± 0.52mg and in the fifth group 138 ± 0.14mg/100g, which indicates an increased sodium content in the eggs where used 5% V + FM feed additive. The content of potassium is also very high, in the eggs of the experimental group 3 it is 154 ± 0.42 mg/100g, which is 18 mg more than the control group, and in group 2 it is slightly higher and is 140 ± 0.32mg. The content of calcium in the eggs of the second and third experimental groups is comparatively higher than in the control group and is 58 ± 0.91 mg and 61 ± 0,13mg, respectively. The magnesium content is also higher in the eggs of the experimental groups of laying hens. The content of phosphorus and iron in the eggs of experimental groups 4 and 5 were 226 ± 0.18; 235 ± 0.01mg/100 g and 3.6 ± 0.03; 3.9 ± 0.62mg/100g, and in the eggs of the control group 185 ± 0.65 and 2.3 ± 0.98mg/100g. It was found that the quantitative content of trace elements in the eggs of the experimental groups is higher than in the eggs of the control group, especially where the hens received vermiculite 3 and 5%. The amount of iron in the third experimental group was 3.9 ± 0.91 mg, its value in the control group was 2,3 ± 0.98mg/100g. The amount of iodine in all groups was in the range of 0.22-0.23 mg. Zinc is found in the eggs of the control group 1.2 mg/100g, only in the fifth group its amount increased by 1.4 mg/100g, where used a feed additive of vermiculite with fish meal. Thus, our research established that the macro and microelement composition of the eggs of experimental groups is the richest in the following minerals: sodium, potassium, calcium, magnesium, phosphorus, iron, zinc. In the control group, their content is lower than in the experimental group. This indicates that vermiculite and feed additives based on vermiculite are the most valuable in nutrition, since they provide the body with minerals.

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3.8 Morphological and histological changes of broiler chickens` meat while using vermiculite in feed Figure 22 shows the transverse and longitudinal sections of the broiler chickens`s meat tissue of experimental and control groups. Smaller in diameter muscle fibers in the tibia of the chicken-broilers of the experimental group give the meat a tender consistency. Broiler meat is classified as a dietary product, with a high level of digestibility (Appendixe S).

a) transverse sections of group E of the broiler chickens`s meat

b) longitudinal sections of group E of the broiler chickens`s meat

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c) transverse sections of control group of the broiler chickens`s meat

d) longitudinal sections of control group of the broiler chickens`s meat

Figure 22– The transverse and longitudinal sections of the broiler chickens`s meat tissue of experimental (a, b) and control (c, d) groups. Stained with hematoxylin-eosin.10x40

Skeletal muscle of chicken`s meat is presented with striated muscle tissue. On longitudinal sections of the muscle fibers were visible shell fibers having the form of the contour line. Under the sarcolemma at the periphery of the nucleus fibers were elongated with small clumps of chromatin. The central part of muscle tissue was occupied by myofibril fibers which had longitudinal striations, stand-out differently in different fibers. The spaces between the striated muscle fibers were filled with layers of loose connective tissue called endomysium.

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When studying the histological sections on the muscles of the experimental and control groups, it was established that the pattern of muscle bands was clearly expressed. In the fields of vision the single hypertrophied muscle fibbers were observed. In the interstitial connective tissue fatty vacuoles were single, their number was insignificant. So muscle tissue of broiler chickens in both control and experimental groups had the same histological and morphological properties [224]. This research provides the first comprehensive demonstration of vermiculite from Kazakhstan as feed additive on broilers performance and meat quality. Vermiculite was not investigated so far as a feed additive and had no toxicity and beneficially influenced on fishmeal. The presence of macro and microelements in vermiculite composition in a sufficiently large amount distinguishes it from other natural minerals.

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The economic efficiency of using vermiculite in feed production and poultry farming High rates of reproduction, feed payment, profitability and return on investment distinguish poultry from other livestock sectors. The bird has a high energy of growth. For 1 kg of gain, it needs 2.0-2.3 kg of mixed fodder. The cost of production is one of the most important economic indicators of the poultry industry. To carry out calculations to determine the actual economic efficiency of vermiculite use in poultry farming, in production-experimental conditions, it is carried out by comparing the test results with vermiculite and without using vermiculite (control and experimental groups of birds). The system of indicators makes it possible to give an economic assessment of the use of domestic vermiculite in poultry farming, thereby comparing the economic efficiency of the mineral used in poultry farming as a feed additive. When carrying out production-experimental tests of natural minerals in poultry farming, it is necessary to follow the norms of the additive and to mixed fodders, since they do not contain nutrients and higher doses of the natural mineral reduce the nutritional value of the feed, and low doses reduce its economic efficiency. onsidering that the mixture of the mineral with fish meal prepared in mixers directly into halls (rooms) before being fed into the feed or smokers thus entered control of feed entering the trough birds. Samples of feed mixtures were taken at least twice a week, directly from the feeders of cellular batteries at 5-8 points with a total mass of not less than 100-200g. The production-experimental experience is one of the most reliable methods for assessing the effectiveness of the use of feed additives. In this case, all of the factors, except the testing, must be comparable. The cost of production, the expenses of its production and net income are the basis for calculating the economic efficiency of the use of natural minerals in poultry farming. The cost of the total and additionally received products from the use of vermiculite is determined by the actual prices of sales of the products. For comparison, in different price zones, products are valued at comparable prices. Net income and profitability of the use of natural mineral are determined by the difference in the cost of production and costs when using vermiculite without them. The most stable and objective indicators of the effectiveness of vermiculite are obtained by analyzing data on a different food background and conditions of detention. The economic efficiency of the use of vermiculite in poultry farming was calculated according to the following system of indicators: increase in the productivity of birds and the size of supplementary products (increment) in natural and value terms, per head and 1 tenge of costs associated with the use of vermiculite, providing the effect; decrease in feed consumption per unit of additionally received and all produced poultry products; safety of livestock,%; Net income (cost of production less costs), obtained as a result of application of vermiculite per 1 head; profitability of production, application of vermiculite,%; In the structure of the cost of eggs and poultry, 70% or more of the expenses falls on the share of feed. At existing prices for purchased feed, the specific weight of protein components is from 25 to 40% of the total cost of the ration. This indicates

118 the need to reduce the cost of the protein part of the feed mixture. The cost of fishmeal using vermiculite varies and, accordingly, the components are changed. The cost of fish meal with 60% protein during the test cost - 315tg / kg. The cost of vermiculite per ton is - 7000 tons, i.e. kilogram of vermiculite costs - 7 tenge. A kilogram of fishmeal with the use of 30% vermiculite cost 222.6 tenge. From these data it can be seen that the use of vermiculite during manufacturing reduces their cost by 92.4 kg / kg. The economic efficiency of the application of vermiculite from the Kulantau field without the feeding of broiler chickens was determined in the experimental production conditions of the poultry farm LLP Sary Bulak (Appendixe T). Two groups of broiler chickens were formed according to the principle of analogues of 100 heads each. In the control group, the chickens received mixed feeds according to the established technology, and in the experimental - feed, with 5% vermiculite added by the weight of the feed. The duration of the experiment was 42 days. The calculation of economic efficiency was carried out by comparing the results of growing chicken broilers in the basic and proposed versions (table38).

Table 38 – Economical effectiveness of using 5% vermiculite supplementation in broiler chickens feed

Index Options basic proposed Number of goals 100 100 Duration of the day 42 42 Average daily gain, g 46,6 51,3 Weight gain, kg 2007,3 2204,2 The cost of one kg of growth 269 228 Additional increase, g - 197 Cost of additional products, kz - 45 The economic effect per head per - 1,07 day, kz

The scientific and production audit showed the effectiveness of using 5% of the Kulantau vermiculite additive in the feeding of broiler chickens during the growing period. The economic effect per head per day was 1,07 kz.

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4 GENERALIZATION AND EVALUATION OF RESEARCH RESULTS

Veterinary and sanitary requirements for the production of vermiculite for feed preparation include the solution of a set of issues that ensure the further harmlessness of the mineral for the organism of farm animals and birds. Natural aluminosilicate vermiculite has a high adsorption capacity, low bulk density and is rich in macro- and microelements (iron, calcium, silicon, aluminum, copper, zinc) and can be used as fillers in the preparation of dry animal feeds. Vermiculite has a high sorption property with respect to fat, its fat content is 52 grams of mineral weight. Technologically quality (caking, flowability, acidic chill, total microbial number) of fishmeal with the addition of vermiculite improves. Vermiculite is an excellent antiserumant and an antioxidant for fish meal. Vermiculite is a new material for the Kazakhstan market, although it is widely used in other countries. Due to its physico-chemical, ion-exchange and sorption properties, vermiculite is a biologically active agent for increasing productivity and natural resistance, preventing diseases and toxicoses, and improving the quality of the end products of poultry farming. The laboratory animals of all the experimental groups, which were added to the diet of vermiculite, had a brilliant appearance, elastic skin, were mobile. By the end of the experience, no cases of lethality have been established. Vermiculite reduces residual amounts of pesticides in feed and feed additives. Introduction to the diet of fishmeal with vermiculite enriches the feed with mineral elements (iron, silicon, calcium, copper, zinc, etc.), does not worsen its amino acid composition and digestibility. Feeding of broiler chickens by vermiculite for 42 days acts positively on many of the physiological parameters of their body. The number of erythrocytes in experienced group chicks was higher than in the control group by 5.5%, hemoglobin by 7.6% and hematocrit by 5.9%. And the feeding of vermiculite to laying hens for 60 days confirmed the positive effect of this aluminosilicate on their organism. The amount of hemoglobin in the experimental group in laying hens increased by 14%, erythrocytes by 2.4%, where 5% of vermiculite was used together with the basic diet as compared with the control group. The amount of calcium in the blood serum of laying hens of the experimental group increased by 37%. Experimental studies show that the use of vermiculite in the diet of broiler chickens from 7-day age, in an amount of 5% of the dry matter of feed, leads to an increase in the growth of live weight by 8.9%, the preservation of livestock by 2%, a decrease in feed costs per unit of production by 8.9%. The use of vermiculite has a pronounced effect on the commodity and technological qualities of the product. The weight of gutted carcass of experienced chickens increases by 3.3%, the mass of pectoral muscles by 5.2%. In addition, physiologically justified an increase of muscular stomach in experimental chickens at 9%. A tasting evaluation showed an improvement in the taste of meat. Estimation of the broth, boiled and roasted meat of the experienced group chickens

120 was 2.1-3.4% higher than in the control groups; 3.1-3.6%; 1.9-2.1% respectively. The broth of the experimental group was more rich, and the meat was juicy and tender. The study of the chemical composition of the pectoral muscles of chickens showed that the meat of the experimental group, where vermiculite and fishmeal was used, has the greatest nutritional value. Protein content by 3-3.8%, dry matter by 3- 4.9% was higher in comparison with control groups, respectively, which increased the energy value by 0.84-3.8%. The high quality of chicken meat from the experimental group is also confirmed by the calculation of the OPF. The difference in the OPF of the experimental group for pectoral muscles was 0.2% and 0.7%, compared with the control groups. The physico-chemical characteristics of chicken meat from the experimental and control groups did not differ significantly. The pH of the meat was 6.9 ± 0.02. The reaction with copper sulfate and Nessler reagent was negative, the reaction to peroxidase was positive, which corresponded to the indicators of fresh and benign meat. The total microbial contamination of the pectoral muscles did not exceed the established norms and was 1 × 102-1 × 103 CFU / g QMAFAnM. The presence of pathogenic microflora was not revealed. The inclusion of vermiculite in the ration of the bird contributes to an increase in egg production by 22% and a reduction in the fight of eggs by 5-8%. Vermiculite exerts a pronounced effect on the physical parameters of eggs: an increase in mass by 2.3%, thickening of the shell by 15%, egg density increases by 9.7%. Amino acid composition of eggs and meat does not deteriorate, their mineral composition improves. The level of vitamins in meat and eggs under the influence of vermiculite did not decrease. It remains at the physiological level. The effect of vermiculite on the internal organs in terms of histology showed that the kidney and liver of test birds are less susceptible to viruses. The organs have a tendency to self-recovery. The spleen of experienced chickens had larger follicles, the intestines of experienced birds differ from control birds. The results of histological studies of carcass meat in both groups showed that the ratio of connective and muscular tissue varies in different parts of the carcass, and the components of the muscle tissue have different size and qualitative composition. The meat of the control and experimental groups in the histological structure (fiber size, thickness of the sarcolemma, the degree of development of the connective tissue) was a uniform distribution of fat between the muscle beams, fine-grained, and also a small number of connective tissue formations between the muscle bundles. The economic efficiency of the use of vermiculite (5% of the weight of the main diet), when added to fishmeal and fed to broiler chickens in the scientific-economic experience was 10.7 tenge per day per 10 animals. Analyzing the data of the research work, it can be concluded that the use of natural mineral vermiculite in poultry farming, veterinary medicine and environmental protection effectively affects to the safety, productivity and quality of poultry products.

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CONCLUSION

1. Veterinary and sanitary research of the vermiculite of the Kulantau field and the plant located in the South-Kazakhstan region allows us to conclude that the plant is equipped with advanced technology. For veterinary purposes, vermiculite grade M150 with particle sizes of 0.6-5.0 mm is used which has a stable chemical composition with 17% silicon, 2.3% calcium, 20.6% iron, 6.3% aluminum, 6.4% magnesium and 8,1 % potassium and others. In electron microscopic studies, a complex microstructure of vermiculite was noted; their pores were formed by microcrystals and aggregates. And it corresponds to veterinary-sanitary requirements for feed preparation. 2. Veterinary-toxicological assessment of vermiculite on laboratory animals showed that this mineral does not have toxicity. In the experiment on white mice, it was found that when using different concentrations of vermiculite in the composition of mice feeds, no toxic effect was detected and it can be attributed to the group of nontoxic drugs. 3. The addition of vermiculite in an amount of 20-30% (by weight) to fish meal, during its storage and preparing, contributes to the improvement of physicochemical and technological properties - reducing the percentage of acid number of fat to 22%, reducing the moisture percentage by 45%, remaining stable during storage. Vermiculite is reduced caking of fish meal and improved flowability. 4. Experimental studies of the level of residues of pesticides in feed showed that expanded vermiculite, due to its unique sorption properties, reduces the content of residual amounts of pesticides when used in the composition of feed additives. Significant decrease in the residual amount of diuron was observed with 30% vermiculite as filler to the mass of fishmeal in the experiment. 5. Growing of broiler chickens on the basic diet of 5% vermiculite makes it possible to obtain an average daily gain of 51g (control group 46.6g), with the use of expanded vermiculite in an amount of 5% of the dry matter of the diet, the gain is increased by 9.1%. The control weighing of birds in the pre-slaughter period where used 5% vermiculite with fishmeal showed an increase of the live weight in the experimental group by 3.6% compared to the control value. The postslaughter weight increased by 9-10 %, slaughter yield by 2%. 6. Examination of the quality of demolished hens' experimental eggs showed a significant increase in the mass of eggs in experimental batches during all periods of oviposition. From laying hens kept on a diet with 5% vermiculite with fishmeal, eggs were obtained, with an average weight of 63.31 ± 0.30 g, which is significantly higher by 2.3% than the weight of control group of eggs. Measurement of the thickness of shell revealed significant differences between control and experimental samples of laying hen eggs, which received 5% vermiculite along with the diet, the shell thickness was 15.3% and the density was 9.7% higher. Similarly, an analysis of indicators that indirectly characterize the strength of the shell (elastic deformation - ED) reliably indicates a high physical quality of the shell in eggs obtained from layers from experimental groups. Thus, in control, the ED was 26.9 ± 0.91 mm, and

122 the experimental was in the range of 19.5 ± 1.01 mm. In production conditions, these indicators become economic factors. These facts allow us to regard vermiculite as a preventive measure of preserving the production marriage - the battle of eggs, and increasing the efficiency of production of table eggs. 7. The introduction of vermiculite to the main diet of birds did not adversely affect the veterinary and sanitary characteristics of meat and eggs, such products for a complex of organoleptic, bacteriological and physico-chemical research methods are benign, suitable for use on food purposes and are subject to free implementation. 8. The addition of 3 and 5% vermiculite to the diet of broiler chickens promoted an increase an improvement in hematological indices, hemoglobin by 4.1%. Hematologic and biochemical indicators corresponded to physiological norms, which indicate the non-toxicity of a natural mineral in the process of feeding birds. 9. Veterinary and sanitary assessment of broilers’ meat who received 5% vermiculite with fish meal to the main ration showed that the chemical composition of meat on protein content is higher by 3.8%, and the amount of fat in the test group where only 5% of vermiculite was used was reduced to 3.3%, this is also explained by the adsorption capacity of vermiculite. 10. When vermiculite used in an amount of 3 and 5% of the main weight of mixed fodders, was included in the body's mineral metabolism of chickens. Thus, muscle tissue in control carcasses contained more such chemical elements as iron by 9.1%, manganese by 2.9%, copper by 1.7%, potassium by an average of 5.3%, in comparison with the experimental samples of poultry meat.

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PRACTICAL PROPOSALS

1. To improve the technological and physical chemical properties of fishmeal, it is recommended to use natural aluminosilicate - vermiculite (20-30% vermiculite to the mass of fishmeal) 2. It is recommended to add 5% of expanded vermiculite (based on the weight of the basal diet) to the diet of laying hens, to enrich the food with macro-microelements (iron, copper, magnesium, silicon, etc.), and to improve the physiological condition of laying hens and biophysical properties of eggs. 3. To increase the resistance, preservation and productivity of the bird, it is recommended to use expanded vermiculite in doses of 3-5% into the basal diet of broiler chickens.

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