Kazakh National agrarian university

UDC: 636.087.63:639.2 On the rights of manuscript

PARITOVA ASSEL ERZHANOVNA

Veterinary-sanitary evaluation of the quality fish while using non- traditional feed additives «Tseofish»

6D120200-Veterinary sanitation

A dissertation submitted for the degree of Doctor of Philosophy (PhD)

The domestic scientific adviser doctor of veterinary sciences, professor Sarsembayeva N.B.; Foreign scientific adviser doctor of chemical sciences, professor of Institute Plant protection (Poland) Lozowicka B.

The Republic of Kazakhstan Almaty, 2014 1

CONTENTS NORMATIVE LINKS 4 DEFINITIONS 6 DESIGNATIONS AND ABBREVIATIONS 8 INTRODUCTION 10 1 REVIEW OF LITERATURE 14 1.1 Veterinary-sanitary examination of fishery products 14 1.2 Aquaculture development in Kazakhstan and actual problems 18 1.3 Feed and feed additives in aquaculture 20 1.4 Zeolites are natural minerals used in veterinary science 23 1.5 The chemical composition and nutritional value of fish 30 1.6 Pesticide residues in feed and fish 35 2 OWN RESEARCHES 38 2.1 Materials and methods 38 2.1.1 Research materials 38 2.1.2 Research methods 41 2.1.2.1 Organoleptic methods 41 2.1.2.2 Methods of biochemical research 42 2.1.2.3 Methods for determining of chemical composition of fish 43 2.1.2.4 Method for determining the amino acid composition of fish 43 2.1.2.5 Methods of determining fatty acid composition of fish 43 2.1.2.6 Method for determining the mineral composition of fish 44 2.1.2.7 Method for determining the vitamin composition of fish 44 2.1.2.7.1 Equipment 44 2.1.2.7.2 Reagents and solutions 44 2.1.2.7.3 Samples and reference materials 44 2.1.2.7.4 Sample preparation 45 2.1.2.7.5 Chromatographic conditions 45 2.1.2.8. Methods of histological examination of meat 45 2.1.2.8.1 Preparing a mixture of egg white with glycerol and processing 45 microscope slides 2.1.2.8.2 Preparing of eosin solution 46 2.1.2.8.3 Preparation of Ehrlich hematoxylin 46 2.1.2.8.4 Staining of slices 46 2.1.2.9 Hematological study of fish 46 2.1.2.10 Determination of pesticide residues in feed and fish meat 48 2.1.2.10.1 Samples and reagents 48 2.1.2.10.2 Standarts 48 2.1.2.10.3 Sample preparation 48 2.1.2.10.4 Instrumentation and chromatographic conditions 48 2.1.2.10.5 Method of validation 49 2.1.2.11 Statistical processing of results 50 3 RESEARCH RESULTS 51

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3.1 Physico-chemical properties of non-traditional feed additive Tseofish 51 3.2 Toxicological evaluation of non-traditional feed additive Tseofish 55 3.3 Study of the effect of non-traditional feed additive Tseofish at fish- 56 biological and hematological parameters of the body of fish 3.3.1 Fish breeding and biological indicators of valuable fish species when 56 using nontraditional feed additive Tseofish 3.3.2 Hematologic characteristics of valuable fish species when using a feed 58 additive Tseofish 3.4Veterinary and sanitary assessment of the quality of fish, when used in 63 the composition of the feed additive feed Tseofish 3.4.1 Organoleptic characteristics and indicators of freshness of fish meat in 63 the diet added feed additive Tseofish 3.4.2 The chemical composition and nutritional value of fish meat when 70 using a feed additive Tseofish 3.4.3 Amino acid composition of fish meat in the diet when using NFA 75 Tseofish 3.4.4 Analysis of the fatty acid composition of fish meat when added to feed 81 NFA Tseofish 3.4.5 Vitamin content of fish meat of valuable species when used in feed 93 NFA Tseofish 3.4.6 Mineral content of fish meat in the application of NFA Tseofish 97 3.5 Morphological changes of valuable species of fish meat using NFA 100 Tseofish 3.6 Study of the content of pesticide residues in feed and fish 102 3.6.1 Pesticide residues in fish feeds when using nontraditional feed additive 117 Tseofish 3.6.2 Pesticide residues in fish when using in feed nontraditional feed 102 additive Tseofish 4 GENERALIZATION AND EVALUATION OF RESEARCH 120 RESULTS 5 CONCLUSION 123 6 PRACTICAL PROPOSALS 125 7 REFERENCES 126 Application A Pesticide residue chromatograms Application B The electron fotos of zeolite crystals Application C Act of research results in the learning process

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NORMATIVE LINKS

In this dissertation references are used to the following standards:

ST RK 1802-2008 Fish, seafood and products their processing. Acceptance rules and sampling. ST RK 1803-2008 Fish and seafood. Sensory evaluation methods. GOST 7631-85 Fish, marine mammals, marine invertebrates and products their processing. Acceptance rules, organoleptic methods of quality evaluation, sampling methods for laboratory testing. GOST 23042-86 Meat and meat products. Method for determination of total fat content. GOST R 51479-99 Meat and meat products. Method for determination moisture mass fraction. GOST 23392-78 Meat. Methods for chemical and microbiological analysis of freshness. GOST 19496-93 Meat. Method of histological analysis. GOST R 53642-2009 Meat and meat products. Method for determination of total ash mass fraction. GOST 25011-81 Meat and meat products. Methods for determination of protein. ST RK 2010-2011 Determination of organochlorine pesticides by chromatographic methods. ISO 24333-2009 Grain and grain products. Sampling. GOST 25336-82 Laboratory glassware and equipments. Types, basic parameters and dimensions. GOST 13496.20-87 Fodder and feed raw materials. Method for the determination of pesticide residues. The Council Directive Veterinary and sanitary requirements for aquaculture 2006/88/ES from animals and their products, and on the prevention and October 24, 2006 control defined diseases in aquaculture animals. GOST R 52346-2005 Combined feed for fish. Nomenclature of indices. ISO/IEC 17025:2005 General requirements for the competence of testing and calibration laboratories. ISO/TC 34/SC 6 Meat, poultry, fish, eggs and their products. ISO 936:1998 Meat and meat products - Determination of total ash. ISO 1442:1997 Meat and meat products - Determination of moisture content (Reference method). ISO 12877:2011 Traceability of finfish products. Specification on the information to be recorded in farmed finfish distribution chains. CAC/MRL 1 Maximum Residue Limits (MRLs) for Pesticides. 4

CAC/GL 55-2005 Guidelines for Vitamin and Mineral Food Supplements. CAC/GL 33-1999 Recommended Methods of Sampling for Pesticide Residues for the Determination of Compliance with MRLs. CAC/RCP 52-2003 Code of Practice for Fish and Fishery Products. CAC/GL 31-1999 Guidelines for the Sensory Evaluation of Fish and Shellfish in Laboratories. GOST R 55483-2013 Meat and meat products. Determination of fatty acid composition by gas chromatography. GOST R 55482-2013 Meat and meat products. Method for determination of water-soluble vitamins.

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DESIGNATIONS AND ABBREVIATIONS

ADIs- Acceptable Daily Intakes ARfDs -Acute Reference Doses ASPM -Agreement about Sanitary and Phytosanitary measures ATP -Adenosine triphosphate BD - basic diet DDT - 4,4 '-dichlorodiphenyl-trichloroethane E.coli - Esherichia coli ECD - electron capture detector EFA - essential fatty acids FA- fatty acid FAME - fatty acid methyl ester FAO -Food and Agriculture Organization GOST - government standard GC - a gas chromatograph g - gram g/l - gram per litre h - hour HAC- Higher Attestation Comission HCH - hexachlorocyclohexane EU - European Union EUPT - European Commission’s Proficiency Testing Program ISO - The International Organization of Standartization IUCN - The International Union for Conservation of Nature kg - kilogram lb - the pound-mass LD - lethal dose LIA - lioneic acid LOD - limit of detection LOQ - limit of quantification mcl - microliter mg - milligram mg/kg- milligram/kilogram ml- milliliter mm - millimeter min - minute MPC - maximum permissible concentration MRL - maximum permissible level MYA - myristic acid ND - normative documents NFA - nontraditional feed additive NPD - the nitrogen-phosphorus detector OCs- organochlorinies OLA- oleic acid 6

PAA- palmitic acid PUFA - polyunsaturated fatty acid RfD – Reference Dose RSD - relative standard deviation S/N - signal-to-noise ratios SRI - Scientific Research Institute STA- stearic acid ST RK - Standard of the Republic of Kazakhstan UK - United Kingdom USA- United States of America WTO - World Trade Organization

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DEFINITIONS

This dissertation uses the following terms with corresponding definitions:

Adsorption - a process that occurs when a gas or liquid solute accumulates on the surface of a solid or a liquid (adsorbent), forming a molecular or atomic film (the adsorbate). It is different from absorption, in which a substance diffuses into a liquid or solid to form a solution. The term sorption encompasses both processes, while desorptionis the reverse process. A fish - is any member of a paraphyletic group of organisms that consist of all gill-bearing aquatic craniate animals that lack limbs with digits. Aluminosilicates - are minerals composed of aluminium, silicon, and oxygen, plus countercations. They are a major component of kaolin and other clay minerals. A mineral - is a naturally occurring substance that is solid and stable at room temperature, representable by a chemical formula, usually abiogenic, and has an ordered atomic structure. 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. A vitamin - is an organic compound required by an organism as a vital nutrient in limited amounts. An organic chemical compound (or related set of compounds) is called a vitamin when it cannot be synthesized in sufficient quantities by an organism, and must be obtained from the diet. Chromatography - (kroʊməˈtɒɡrəfi/; from Greek χρῶμα chroma "color" and γράφειν graphein "to write") is the collective term for a set of laboratory techniques for the separation of mixtures. The mixture is dissolved in a fluid called the mobile phase, which carries it through a structure holding another material called the stationary phase. Clinoptilolite - is a natural zeolite comprising a microporous arrangement of silica and alumina tetrahedra. It has the complex formula: (Na,K,Ca)2- 3Al3(Al,Si)2Si13O36·12H2O. It forms as white to reddish tabular monoclinic tectosilicate crystals with a Mohs hardness of 3.5 to 4 and a specific gravity of 2.1 to 2.2. 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. Fisheries - branch of national economy engaged in the catch, transport, treatment, cultivation and protection of fish and aquatic plants. Fish products - fish in natural or processed form, intended for use for food purposes. 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

8 processes of purification, separation, and decontamination of aqueous and other ion-containing solutions with solid polymeric or mineralic 'ion exchangers'. Live fish - fish swimming in a natural or approximated habitat with the natural movements of the body, the jaws and gill covers. Pesticides - are substances meant for attracting, seducing, destroying, or mitigating any pest. 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 rainbow trout - (Oncorhynchus mykiss) is a species of salmonid native to cold-water tributaries of the Pacific Ocean in Asia and North America. The sterlet - (Acipenser ruthenus) is a relatively small species of sturgeon from Eurasia native to large rivers that flow into the Black Sea, Azov Sea, and Caspian Sea, as well as rivers in Siberia as far east as Yenisei. Underyearlings - this summer (year) fish. Zeolites - are microporous, aluminosilicate minerals commonly used as commercial adsorbents and catalysts.

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INTRODUCTION

Topicality. Kazakhstan has a rich fish agriculture water resource and favourable conditions for the intensive development of fish culture and fisheries. Considering the predicted increase in the population of the republic, and proceeding from norms recommended by science (14,6 kg per capita) to satisfy the needs of a population for fish and fish production, it is necessary to increase the volume of fishing-out, cultivation of commodity fish and the import of fish to 272 thousand tons a year [1]. Currently, one of the main problems of the agro-industrial complex is meeting the needs of the population for fishery products of the required range, of high quality and at affordable prices, which is impossible without an increase in the productivity of valuable fish species [2]. The fishing industry is one of the leading sectors of modern agriculture. The bulk of the catch of fish is derived from the oceans and seas, but, along with commercial fishing the breeding and rearing of fish in inland waters - ponds, lakes, and reservoirs is also imported. With intensive pond culture many fish products can be produced from small areas of water. Fish is one of the staples in the diet of persons of any age, providing an irreplaceable source of complete protein, fats, vitamins, minerals and other vital nutrients [3]. One of the reasons that prevent fish farming and the breeding of fish in fish farms, as well as the low quality of fish caught in natural waters, are unbalanced feeding, and a lack of quality and affordability of feed additives [3-4]. Feeds during their production process undergo a series of anthropogenic and biological pollution [5-7]. The reduction and elimination of the negative impacts of toxins and pesticides in feed on fish allow inclusion in the diet of sorbents of natural origin which can increase the average daily growth of fish by 6,5%, reducing the cost of feed per unit of production by 13,0% and also increase the quality of the fish as a food[8-10]. In recent years, scientists have paid great attention to the use of non- traditional feeding fish feed additives (zeolites, bentonite, etc.)[11]. Early work testing zeolites in fish-farming showed the possibility of their successful application in the form of feed additives [32-33]. There is a reason to hope that the use of zeolites and other aluminosilicate zeolite geylendit-series can have a significant positive effect on the feeding of fish. Previous studies in this area can be characterized as fragmented and not giving enough information to recommend the use of zeolites in the tuffaceous composition of fish feed. In this regard, we have carried out a study whose goal was to determine the effectiveness of using zeolite as an additive in the feed for selected fish [12]. The basic method of the intensification of the production of pond fish, which allows significant increases in the yields of fish production per unit of water area, is the feeding of the fish [13]. Along with improving the fodder base, with an increase in the production of various feeds, it is necessary to improve the usefulness of diets by enrichment of their complex feed additives. Given the 10 scarcity and high cost of imported mineral and, biologically active substances, it is necessary to develop feed additives based on existing natural resources in the Republic of Kazakhstan. Using mineral additives based on local zeolites have been studied and have been shown to be positive for livestock and poultry, but for fish there is almost no extant work [14-17]. Currently, in intensive cultivation conditions, environmental contamination with waste from industrial plants cause accumulation of heavy metals, pesticide residues, herbicides and other toxins in feed and feed additives. Therefore, an innovative trend is the application feeding fish different sorbents that, due to their chemical composition and physical and mechanical properties, possess unique ion exchange and adsorption properties, and are involved in various exchange processes. They can correct of the biochemical and antigenically structural homeostasis of the animals’ metabolism, allowing the production of organic food [18]. However, it should be remembered that a significant risk for fishery ponds is contamination by pesticides and the destruction of fish by infectious and non- communicable diseases [3]. Questions have not been studied regarding the influence of mineral additives on the veterinary and sanitary evaluation of fish meat, nor on the influence of zeolites on organic supertoxicants in fish, despite the fact that pesticides such as HCH, DDT, and heptachlor are very dangerous pollutants; they cause suppression of cellular immunity leading to liver disease, and damage to the generative organs, etc. [19-21]. Therefore, according to veterinary legislation, fish, crabs and other aquatic organisms, fished for the purposes of food and animal feed, regardless of epizootic state fishery ponds, must be subjected to animal health inspection at the point of harvest. Prohibited from use in human food are fish containing residues of the following pesticides: aldrin, afugana, heptachlor, dinitroortokrizola, dihloralmocheviny, Metaphos, nitrofen, arsenic preparations thiophos, yellow and white phosphorus and, mercury-containing products. Sturgeon fish (sturgeon, sterlet, beluga, stellate, spike) are not subject to Veterinary-sanitary expertise in markets [22]. The purpose and research problems. The purpose of this dissertational work was the veterinary-sanitary evaluation of the quality and safety of fishery products when the untraditional feed additive Tseofish is used in the composition of the feed. To accomplish this goal the following tasks were set: 1. Investigate the technology of producing the non-traditional feed additive Tseofish to develop veterinary and sanitary requirements for its production. 2. Establish the levels of pesticides in feed and the feed additive Tseofish for feeding valuable species fish. 3. Investigate the effects of different doses of the non-traditional feed additive Tseofish on the test organism the guppy fish (Poecilia reticulata).

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4. Provide a veterinary and sanitary evaluation of the quality, safety and nutritional value of fish meat when using the non-traditional feed additive Tseofish. 5. Investigate the effects of different doses of the non-traditional feed additive Tseofish on the natural resistance of the organism when used in the fish diet feed additive Tseofish. 6. To study morphological and histological changes in the fish meat when using the feed additive feed Tseofish in the diet composition. 7. Determine the level of pesticide residues in the meat of fish when the feed additive Tseofish was used in their diet. Scientific novelty of the research results. Veterinary and sanitary requirements were developed which are applicable to non-traditional technology of production of the feed additive Tseofish. The peculiarities of the effect of various doses of the feed additive Tseofish on the natural resistance of the organism and the morphology of the internal organs of fish were investigated. Veterinary and sanitary evaluation of fish meat was made using the non- traditional feed additive Tseofish based on a comprehensive experimental study. The physicochemical properties and chemical composition of fish meat were explored, given the morphological and histological evaluation of muscle tissue and internal organs of fish using this feed additive. It has been shown that the feed additive Tseofish while not altering the veterinary and sanitary parameters and safety parameters of fish meat, reduced the level of pesticide residues in feed and in the flesh of the fish. The practical value of the work. The mechanism of the influence of the non-traditional feed additive Tseofish on veterinary-sanitary evaluation and quality of fish, and its safety, was studied. The possibility of enriching fish meat proteins, amino acids, vitamins, and macro-micronutrients when the feed additive Tseofish was used in the composition of fish feed was determined. The results of these experimental studies can be used in education for veterinary and sanitary examination, morphology, and biochemistry subjects. The research conducted allow us to recommend the use of Tseofish in fish farming in order to increase the safety of fish, meat productivity, enhance the nutritional and biological value of fish meat, adjusting the mineral and protein metabolism of fish. Research results are used in the education of students of the Kazakh National Agrarian University, the West Kazakhstan Agro-Technical University named after Zhangir Khan, Irkutsk Agricultural University, and the Warmino Mazury University in Olshtyn (Poland). Approbation of the work. Materials of the thesis and the results of scientific and experimental studies have been presented at international scientific conferences:

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- Materials of 9 of the International scientific-practical conference "Modern Problems of Arts and Sciences", Moscow, 2011; - 66th scientific conference of young scientists and students, St. Petersburg, 2012; - Knowledge of young for Development veterinary medicine and agribusiness country, St. Petersburg, 2012; - Veterinary and Livestock: theory, practice and innovation, dedicated to the 80th anniversary of academician K.Sabdenova. - Almaty, 2012; - International Conference of Latvian agrarian university, Jelgava, 2012; - Materials of the 19th International Scientific and Practical Conference "Veterinary medicine, agriculture, biological and chemical sciences: development in the 21st Century "(London-Odessa), 2012. - Eurasian integration: the role of science and education in the implementation of innovative programs, Uralsk, 2012. - Proceedings of the international scientific-practical conference dedicated to the 70th anniversary of Professor K. Asanova, Almaty, 2012, - International Conference of Latvian agrarian university, Jelgava, 2013. Publication of research results. According to the materials of the dissertation published 21 publications, including four in magazines recommended WAC ("Research results", "Intellect, idea, innovation"), which reflect the main results of experimental studies, 13 - in the proceedings of international conferences, 3 - in the journals, included in the company's database Scopus, 1 - in journals with high impact factor included in the database of Thomson Reuters. Volume and structure of the dissertation. The thesis work was presented to a common pattern. It consists of an introduction, literature review, materials and methods, results of our research, discussion of results, conclusions, list of sources used 136 items. The dissertation is presented on 125 pages of text, drawn up in compliance with the required standards, illustrated with 19 tables and 17 figures.

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1. REVIEW OF LITERATURE 1.1 Veterinary-sanitary examination of fishery products Veterinary-sanitary examination is one of the main and components of veterinary-sanitary sciences, aimed directly at protecting human health [22]. Veterinary-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. According to Kh.S. Goreglyad veterinary-sanitary examination is a scientific discipline that studies the methods of sanitary and hygienic research of food (meat, milk, fish, eggs, honey, etc.) and raw materials (wool, bones, intestines, etc.) of animal origin, as well as some plant products [23]. Veterinary-sanitary examination controls food quality and the quality of raw material of animal origin in their places of production (in livestock farms), at all stages of processing (meat processing plants, dairies, etc.), during transport, storage and at market (bazaars). By monitoring food quality the Veterinary Service takes an active part in improving the health of the people and in protecting them against zoonotic infectious and parasitic diseases transmitted through animal products. It also takes an active part in protecting the public from food-borne diseases caused by poor quality products and products from animals subject to emergency slaughter [24-25]. Veterinary-sanitary examination is a scientific discipline that elaborates methods of research and veterinary-sanitary evaluation of animal products. The term veterinary-sanitary examination was introduced into Kazakhstan in the 20th century. The main practical significance of veterinary-sanitary examination is the prevention of diseases transmitted to humans through food and technical products of animal origin [26]. Veterinary-sanitary examination as a scientific discipline is closely linked with neighbouring sciences such as: microbiology, parasitology, epizootiology, pathological anatomy and histology. Many of the problems of veterinary-sanitary examination are also being addressed by these sciences [27]. Although veterinary- sanitary examination as an independent branch of knowledge emerged only in the 20th century, development of research methods and the rejection of undesirable animal products were practiced much earlier. In the late 19th early 20th centuries, information about veterinary-sanitary examination was part of meat industry practice and, of food hygiene. Recent advances in veterinary-sanitary expertise are related to the development of methods for post-slaughter diagnosis and sanitary evaluation of invasive disease in meat, leukaemia, and pigs’ chronic (localized) anthrax, rapid diagnosis of food poisoning and chemical methods for determining the quality of the meat of sick animals [24]. Veterinary-sanitary examination has currently acquired special importance. Kazakhstan, which entered the path of an open economy, is making steps aimed at further liberalization of activities in the field of foreign trade. In particular, the country's accession to the World Trade Organization (WTO). In this regard, 14 activities aimed at the country's earliest integration into the global food market takes on special significance. The implementation of these measures provides for the creation of a system of harmonized rules and methods to assess the quality and safety of food commodities and products of animal origin and the carrying out of veterinary-sanitary examination and certification in accordance with the requirements "Agreement about Sanitary and Phytosanitary Measures" (ASPM) of the WTO and the FAO standard (World Food organization). In this regard, new, improved and previously adopted normative and legal documents have been recently developed and approved in our country [19]. The rules for the veterinary-sanitary inspection of food products, to determine their safety were approved by the order of the Minister of Agriculture of the Republic of Kazakhstan from April 1, 2008 № 199 (registered in the Register of state registration of regulatory legal acts of the Republic of Kazakhstan 28 April 2008 under number 5198) [28]. The rules have been developed in accordance with the laws of the Republic of Kazakhstan, dated 10 July 2002 "On Veterinary" and dated 21 July 2007 "On the safety of food products". They establish procedures for the veterinary-sanitary inspection of food products at all stages of an animal’s life cycle and apply to all factors involved in the harvesting (slaughter) of animals, production, processing and marketing of food products subject to veterinary supervision [29]. In the rules, veterinary and sanitary expertise means the inspection of the conformity to veterinary norms of animals, products and raw materials of animal origin by means of organoleptic, bio chemical, microbiological, parasitological, toxicological and radiological studies with in the procedures established by the state authorized veterinary body[30]. Veterinary-sanitary examination, as a function of the Veterinary Service, comprises pre-slaughter and post-slaughter diagnosis of animal diseases, and analyses researches of meat, milk, fish, eggs and their products [19]. According to the data presented by K. Malovastyi veterinary and sanitary evaluation of the fish is part of the general veterinary supervision for fish and fishery products. Veterinary-sanitary examination of fish includes: - identification of changes in the studied fish which cause spoiled food or affect the commodity quality, as well as pathogens of diseases transmittable to humans and/or animals (zooanthroponoses and zoonoses); - identification of poisonous fish or fish affected by substances toxic to humans and/or animals; - determination of fish freshness [31]. Fresh fish is a highly perishable product. In the summer it can spoil within 12-24 hours. This is due to the friability of the connective tissue, the negligible content of glycogen, the presence of mucus (mucin) on the body surface, which promote the rapid multiplication of microorganisms, increase the activity of intestinal enzymes that cause tissue lysis, and rupture of the abdomen. In this regard, it may be necessary to establish the degree of freshness of the fish. Moreover, fish may be affected by infectious and parasitic diseases, and maybe exposed to residual

15 amounts of a range of toxic substances, which should also be taken into account at sanitary evaluation [32-34]. For determining the freshness of the fish the whole batch of fish presented for sale or for industrial processing must be inspected. Attention should be paid to the general appearance of the fish; scales and slime condition, colour of the gills, eye condition, abdomen, muscle texture, and smell. Moreover, the microbial contamination of muscle tissue is identified by preparing smears followed by Gram stain – which can reveal the presence of ammonia and hydrogen sulfide. Sample cooking is conducted similarly to that for meat analysis [19]. It is mandatory to carry out a study investigating the presence of helminths. Fish with a bloated abdomen are examined by autopsy to detect ligulosis, abdominal dropsy and other diseases [32]. On examination of live fish attention is paid to their condition within the cages. Healthy fish are mobile and swim at depth. Inactive fish are caught and examined for infectious and invasive diseases. Fish with bruising, or loss of scales are not released for sale, but are sent for industrial processing. The depleted fish direct to utilization [19]. Fish with damaged skin or rapped scales are not released for sale, neither fish with skin lesions. Due to the fact that such fish cannot be stored, they are handed over for processing in fish factories or at public catering establishments, or for processing into animal feed. The depleted fish are also not released for sale, but maybe used for animal feed [35]. Conclusions the good qualities of fresh fish are made on the bases of organoleptic characteristics. Attention should also be paid to the condition of the skin, scales, slime, gills, eyes, abdomen, internal organs, muscular consistency, the presence of fluid in the abdominal cavity, the smell of the mucus, gills and anal area, and also sample cooking is carried out. The whole batch should be visually inspected. At least 30 samples from the batches are subjected to sensory evaluation. Pathoanatomical autopsy must be carried out on 3-5 replicates. For sample cooking 100 g of a purified sample including fish scales, without internal organs, is taken, double the volume of water is added and it is boiled for 5 min. Fresh fish show the following features: shiny scales, scales tight to the body, clear mucus, free from additional smell, the skin is elastic, and tight to the touch, the gills covers are tightly closed to the gill cavity, the eyes are convex or slightly sunken and, the cornea is clear, the abdomen is not swollen, the anal opening is tightly closed, without expression of mucus, the muscle tissue is elastic when cut, and attached tightly to the bones, the internal organs are well defined, the intestine is not distended, without putrid odour. The broth of fresh fish is transparent, with a glitter of fat on the surface, it should smell pleasant. The meat should be well divided into muscular bundles. Fresh, healthy fish are covered with a thin layer of transparent or slightly tarnished mucus. Whole scales, with a shiny pearlescent tint, are firmly attached. The skin of fish without scales is smooth, shiny, slightly tarnished and covered with transparent or slightly tarnished mucus. The eyes should be shiny, bulging or

16 slightly sunken into the orbit. The gills should be pale pink or a vivid red and, covered with mucus, with no signs of decomposition. The muscles should be firm, elastic, and resilient, when pressing on the skin with the finger fossa should not remain. The fish should have a characteristic fresh smell. When sample boiling the broth should be transparent, and smell pleasant [36]. Fish of questionable freshness have the following characteristics: dim scales easily removed, mucus is muddy, sticky, with a sour smell, the skin is easily separated from the muscle, the gill covers are tightly closed, the mucus on the gills is damp and has a musty smell, eyes are sunken and glassy with dim cornea, the abdomen is strained and sometimes bloated the muscle tissue is softened, easily separated into individual fibers, the kidney and liver are in the process of decomposition, with bile staining the surrounding tissue with a yellow-green colour, the intestines are slightly swollen. The broth from such fish is muddy, with little fat on the surface, the smell of the meat and the broth is unpleasant. When the freshness of fish is questionable laboratory analysis should be conducted: bacterioscopy smears of muscle, pH, reaction to hydrogen sulphide, reaction with peroxidase extract from the gills and with Nessler's reagent. Fish of questionable freshness is not suitable for long-term storage. In the case of absence of muscle putrid odour and negative results of laboratory studies the fish can be used in food after heat treatment. When detected in the muscle tissue of Salmonella, E. coli, Proteus fish fed animals after procooking [37]. With a significant colonization fish meat of dubious freshness microorganisms (more than 100 in the field of view of the microscope) it utilized. The scales of substandard fish are folded, the skin is weak and easily separated, the mucus is cloudy with a dirty gray, and it is sticky and foul smelling. The gills are dirty-gray colour, covered with muddy, stringy mucus, and have a foul, putrid odour. The eyes are sunken. Dark or greenish spots can be observed on the surface of the abdomen. The muscle tissue is loose and soft. The internal organs are dirty gray colour, merged into a homogeneous mass, expelling a sharp, putrid smell. Substandard fish broth is highly turbid, and the smell of the meat and the broth is unpleasantly putrid. Poor quality fish kill. Veterinary and sanitary assessment of fish is carried out on the basis of organoleptic, biochemical and microbiological studies. For game fish, they are inspected and for permission to market them, a veterinary certificate must be issued in the form number 1 or a certificate (for sale in the markets of the district), which indicates that the fish have been examined, and the fish and the fish pond are free from infectious diseases and zooanthroponoses [38]. Fish from ponds free of fish diseases and zooanthroponoses and not contaminated with pesticides above permissible concentrations can be marketed after veterinary inspection without restrictions. In all other cases, the fish can be released onto the market after appropriate investigation. Fish recognized as acceptable, can be marketed after clearance that they are free from parasites. When the affected parts of carcasses or parasites are removed such cull fish are checked, so that they do not fall into the pond and do not serve as a source of infection for the other fish. Such fish can not be fed to pets (especially

17 dogs and cats). Fresh meat and entrails, if infested with parasites, are dangerous to the health of humans and animals [39]. Fish proven unsuitable for food, depending on the degree and type of problem are disposed or destroyed. Decontaminated fish should be cooked in open pots at 100°C until such a time when the muscles are not easily separated from the bones. In this case, they can be used in feed for pigs, poultry, dogs or fur animals. Technical recycling of fish must be carried out under the supervision of veterinarians at the processing plants [40]. Regarding the technical and recycling or destruction of fish by a veterinarian is covered by an act indicating recycling process, the type and quantity of technical products received and the number of fish destroyed [40-44].

1.2 Aquaculture development in Kazakhstan and actual problems Kazakhstan, the ninth largest country and the largest landlocked country in the world, is located in Central Asia. The arid and semi-arid nature of much of the country accounts for its relatively low number of rivers, despite its immense size. The country has an estimated 8 500 rivers of note (neighbouring Kyrgyzstan, with a surface area barely one-thirteenth of Kazakhstan, has almost 30 000). There are an estimated 48 000 lakes in Kazakhstan, although a number of them dry up in the hot summer period. The most important lakes are Lake Balkhash, Lake Alakol and Lake Tengiz. The Caspian Sea was, historically, a major source of the global sturgeon catch, but overfishing in recent years has threatened sturgeon stocks to such an extent that severe controls over landings have been introduced so as to support stock recovery. Six sturgeon species inhabit the Caspian Sea basin: Beluga (Huso huso), Starry sturgeon (Acipenser stellatus), Russian sturgeon (A. gueldenstaedtii), Sterlet (A. ruthenus), Persian sturgeon (A. persicus) and Fringebarbel sturgeon (A. nudiventris). Practically all the Caspian sturgeons ascend the Ural River for spawning, with three of them, the Beluga, Russian and Starry sturgeon having high commercial value. A ban on commercial fishing of Fringebarbel sturgeon in the Ural River was imposed in 2002 because there was a very low abundance of these Caspian sturgeon species. There is an exception for catch for restocking and research purposes [45]. The capture fisheries and aquaculture sectors produced some 110 million tonnes of fish for human consumption in 2006. The accompanying apparent per capita supply of 16,7 kg (live weight equivalent) was the highest on record. Aquaculture accounted for 47 percent of the total fish supply worldwide in 2006. In contrast, per capita availability of fish in Kazakhstan was just around 3 kg in 2006 and the aquaculture share of total production was less than 1 percent. Inland capture fisheries and aquaculture sectors in Kazakhstan have been going through a dramatic decline in production, which started after independence in 1991 and lasted until 2001 for capture fisheries and continues until today for aquaculture production. Reasons for the decline are numerous and include, among others, poor water management, reduced state funding, fragmentation of authority over the

18 sector, limited access to fish feeds, unsuitable pond systems, limited policy guidance, and incomplete and obsolete legal frameworks for the sector [46]. The rainbow trout (Oncorhynchus mykiss) is a species of salmonid native to cold-water tributaries of the Pacific Ocean in Asia and North America. The steelhead (sometimes "steelhead trout") is an anadromous (sea-run) form of the coastal rainbow trout (O. m. irideus) or Columbia River redband trout (O. m. gairdneri) that usually returns to fresh water to spawn after living two to three years in the ocean. Freshwater forms that have been introduced into the Great Lakes and migrate into tributaries to spawn are also called steelhead. Adult freshwater stream rainbow trout average between 1 and 5 lb (0,5 and 2,3 kg), while lake-dwelling and anadromous forms may reach 20 lb (9,1 kg). Coloration varies widely based on subspecies, forms and habitat. Adult fish are distinguished by a broad reddish stripe along the lateral line, from gills to the tail, which is most vivid in breeding males. Wild-caught and hatchery-reared forms of this species have been transplanted and introduced for food or sport in at least 45 countries and every continent except Antarctica. Introductions to locations outside their native range in the United States (U.S.), Southern Europe, Australia and South America have damaged native fish species. Introduced populations may impact native species by preying on them, out-competing them, transmitting contagious diseases (such as whirling disease), or hybridizing with closely related species and subspecies, thus reducing genetic purity. Other introductions into waters previously devoid of any fish species or with severely depleted stocks of native fish have created world-class sport fisheries such as the Great Lakes and Wyoming's Firehole River [47]. The rainbow trout live in the river Chilik in Kazakhstan. There are two subspecies of rainbow trout in Kazakhstan – the Caspian and the Aral rainbow trout [48]. Rainbow trout prefer clear, well-oxygenated, cold-water streams with gravel or rocky bottoms, deep pools, and natural cover. Unlike native brook trout, however, they also thrive in large lakes as long as there is cool, deep water. Their ideal temperature range is between 10° and 16° C (50° and 60° F) although they can survive warmer temperatures than some other species of trout. They are more sensitive to acidic water than brook or brown trout and prefer water with a pH of 6 to 8. Stocked rainbow trout can adapt to virtually any waters as long as they have proper habitat, year-round ideal temperatures, and adequate food sources. The sterlet (Acipenser ruthenus) is a relatively small species of sturgeon from Eurasia native to large rivers that flow into the Black Sea, Azov Sea, and Caspian Sea, as well as rivers in Siberia as far east as Yenisei. Populations migrating between fresh and salt water (anadromous) have been extirpated. Due to overfishing (for its flesh, caviar, and isinglass), pollution, and dams, the sterlet has declined throughout its native range and is considered vulnerable by the IUCN. Restocking projects are ongoing, and it has been introduced to some regions outside its native range, but the latter have generally not become self-

19 sustaining. Today, the majority of the international trade involves sterlets from aquaculture [49]. The sterlet is probably the slowest growing and therefore the best species for the garden pond. It only grows to 1,2 m in the wild, but usually up to 1 m in the size average pond also individual specimens can vary in size, sometimes only reaching 60 cm. Conventional weight and length of fishing sturgeon - 0,5-2 kg and 30-65 cm, rarely 3-4 kg and 80-90 cm, as an exception - 6-8 kg. The greatest weight of starlet is 16 kg and length 100-125 cm. Sexual maturity occurs in sterlet males in 3-7 years (mostly 4-5 years) in females - in 5-12 years (mostly 7-9 years) in reaching the length of 28-34 cm. The fecundity of Ob starlet are 6-45 thousand eggs, Irtysh sterlet - 6-16 thousand eggs, Severodvinsk 4-140 thousand eggs. Sterlets spawn after 1-2 years. The males become sexually mature at the Volga River at the age of 3 years, females spawn on the 6th year. There is winter and spring race on the Volga. Sterlet feeds by invertebrates, primarily by insect larvae sitting on sunken driftwood. Chironomid larvae are devolved. Starlet in nature forms a cross between the sturgeon and stellate sturgeon ("sturgeon thorn", "stellate sturgeon thorn"). "Sturgeon spike" is not rare on the Volga; "stellate sturgeon spike" is known on the Volga, Don and the Danube. The hybrid of Siberian sturgeon and Siberian sturgeon (the so-called "Fire") well known in the Ob and Yenisei [50-54]. The value of the fish is the high content of zinc, fluoride, molybdenum and nickel. Vitamin PP and omega 3 fatty acids are very good effect on brain activity and blood circulation, so starlet helps maintain cardiovascular system is normal, and the probability of have a heart attack is greatly reduced, moreover, that the fish reduces the risk of substandard tumors. The same fatty acids, according to experts, contribute to the improvement of vision and even weaken the functions of psoriasis. The high content of fluorine strengthens the skeletal system, and helps for bone growth [55].

1.3 Feed and feed additives used in aquaculture Fish feeds must be formed in to particles or pellets that are strong enough to withstand normal handling and shipping without disintegrating. More-over, fish feeds must be somewhat water-stable. These requirements make it necessary for feeds to contain binders. There are numerous materials that act as binders in fish feed, including regular feed ingredients and ingredients added solely for their binding properties. Some binders are by-products of cereal grains or plants and provide nutrients to the diet. For example, 20% pregelatinized potato starch is added to eel diets to increase the water stability of the dough and to provide energy. Other commonly used binders include bentonite, lignin sulfonate, and hemi cellulose extract, none of which provides nutrients to the diet [56]. Bentonite is naturally occurring clay consisting mainly of trilayered Aluminium silicate. It is available as either sodium bentonite or calcium bentonite. Sodium bentonite has, by definition, more than 1% and less than 2% available ion content,

20 or sodium exchange. It swells when added to water, while calcium bentonite does not. Both sodium and calcium bentonite may be added to dry, compressed fish feeds at no more than 2% to act as a binding agent and also as a lubricant, increasing pellet mill production rates and pellet mill die life. Some bentonites also bind aflatoxin, carrying it through the gut without harming the fish. Lignin sulfonate is a product of the wood pulping industry. It aids in pellet binding, reduces fines, and permits the addition of more steam during the manufacture of compressed pellets. Lignin sulfonate is added at up to 4% as a pelleting aid in dry, compressed (steam-pelleted) feeds. Hemi-cellulose extract is a product made by spray-drying the concentrated, soluble byproduct of pressed wood manufacture. It is less commonly used than lignin sulfonate. Moist and semi moist fish food production requires the use of both nutritive and non nutritive binder materials [57- 60]. Nutritive binders include oat groats, vital wheat gluten, finely milled wheat bran, cottonseed meal, gelatin, fish hydrolyzates, and pre gelatinized starches. Nonnutritive binders include tapioca, carboxymethylcellose, alginates, agar, and various gums. Chitosan, carageenan, and collagen have been evaluated as binders but are not commonly used. Semi moist feeds, containing 25-35% moisture, can often be made into satisfactory pellets by careful selection of feed ingredients that possess binding properties. However, when feed formulations contain ingredients that do not possess suitable binding properties, it is necessary to add ingredients specifically to bind the diet. Moist feeds, having moisture contents of 35 to 70%, always require the addition of a binder. For example, semi purified test diets, such as H440, the Oregon Test Diet, and the Guelph semipurified diet, include gelatin and carboxymethyl cellulose as binders. Moist diets, which are combinations of wet fish ingredients and dry meal, may contain 0.5-2.0% alginates as binders. Heinen found that alginates were better binders than gum, carageenan, chitosan, collagen, carboxymethyl cellulose, and corn starch in a 41% moisture diet. Agar was an effective binder, but expensive. Calcium ions and a sequestrant, such as sodium hexametaphosphate, must be present in diets containing alginatesas binders to control alginate activation [61-63]. A great deal has been written about the addition of carotenoid pigments to fish diets to colour flesh and/or eggs. Over 300 pigments are found in various plants and animals, with xanthophylls and carotenoids being the most important classes of carotenoid pigments that add color to fish. For the most part, xanthophylls are found in plants, such as corn, and carotenoid pigments in crustaceans and fish. Some finfish and shellfish possess the ability to convert certain xanthophyll pigments to carotenoid pigments. Gold fish and common carp can convert the yellow xanthophyll pigment, zeaxanthin, to the red carotenoid pigment, as taxanthin. Similarly, Penaeus japonicus, a shrimp, can convert both β - carotene and zeaxanth into astaxanthin. Salmon, trout, and red sea bream, which normally have pigmented flesh and skin, do not convert xanthophylls pigments to the carotenoids, can thaxanthin, and astaxanthin. In nature, they receive these pigments in their diet. Fish raised in hatcheries and farms must receive

21 canthaxanthin and/or as taxanthin in their diets to become pigmented; in addition, carotenoid supplementation is necessary for salmonid offspring to produce viable offspring. In nature, carotenoid pigments are synthesized by algae and bio concentrated in the food chain, ultimately ending up in fish [64]. Carotenoid supplementation of fish diets is accomplished by adding natural materials containing the desired carotenoid pigments, carotenoid extracts of natural products, or chemically synthesized pigments. Natural materials that pigment fish include herring gull eggs, salmon eggs, paprika, zooplankton, krill products, Haematococcus algae, and processing waste from shrimp, crab, and crayfish processing. Dietary levels of 20% or more of wet crustacean processing waste are required to get the desired pigment in trout and salmon. Concentrated carotenoid extracts of red crab and cray fish are effective dietary supplements for salmonids The amount added to the diet depends on the concentration of carotenoid pigments in the extract, but dietary levels normally range from 3 to 7%, replacing added fats and oils. Synthetic canthaxanthin is a commercial product containing a minimum of 10% canthaxanthin and is added to commercial feeds at 0.05% to produce a dietary canthaxanthin level of about 50 mg/kg feed. Astaxanthin is themost widely used, manufactured carotenoid pigment. It contains 8% asta-xanthin, by weight, encapsulated in gelatin, and is added to fish feeds at approximately 0.065% to produce a dietary astaxanthin level of 45mg/kg feed. Astaxanthin is produced by several microorganisms, including Phyaffia yeast and Haematococcus algae meal, and products are being produced from these microorganisms specifically for use in fish feeds. Because they are produced naturally, they are desirable for use in salmon production for markets demanding a natural food product. Krill products fill the same market niche and are also effective feed palatability enhancers [65]. Rainbow trout are fed formulated feeds throughout farm production cycles and to maturation in the case of broodfish. А. Tacon and M. Metian estimated global production of feed for rainbow trout was 790 000 tonnes in 2006 [66]. Most rainbow trout feed is produced by cooking-extrusion to make floating pellets. Extruded pellets are top-dressed with oil to achieve levels of 18–25 percent total lipid. Fry and fingerling feeds have lower lipid levels than feeds for grower fish [67]. Feed ingredients used in rainbow trout feed formulations are similar throughout the world. Protein sources include fishmeal, poultry by product meal, soybean meal, corn gluten meal, blood meal, feather meal and meat and bone meal. In the case of starter feeds, soy protein concentrate or wheat gluten meal is sometimes used, but other plant proteins are generally not included. Krill meal is also sometimes used if it can be found. Lipids used in trout feeds include fish oil, soy oil and canola oil. A source of starch must be included in extruded trout feeds to act as a nutritional binder. Ground, whole wheat, wheat starch and corn starch are examples of ingredients used in feeds. Vitamin and mineral premixes are also added. If pigmented trout are being produced, astaxanthin is also included. Sufficient information has been obtained by researchers throughout the world to permit feed formulation to be based in part on available nutrient levels in feed

22 ingredients. Hence, most feed producers formulate trout feeds to contain a minimum digestible protein and energy content. Development of models to predict phosphorus availability in formulated feeds is progressing and likely to become a part of feed formulation in the future [66]. Feed additives are added to rainbow trout feeds for a variety of reasons, such as adding microbial phytase to release phosphorus from phytic acid in plant protein concentrates or adding astaxanthin to pigment fillets or eggs in the case of broodfish. Other additives are added to certain feed formulations to enhance feed intake, such as adding betaine to feeds containing rapeseed protein concentrate. Mold inhibitors are generally not added to trout feeds because feeds are typically used soon after production, and rainbow trout farming is generally practiced in areas with relatively cool climates. For feeds produced by compression pelleting, lignin sulfonate is added as a feed binder. Bentonite clay has been shown to reduce levels of aflatoxin, a mold toxin found on grains exposed to high moisture before harvest or during storage [65]. In recent years, feed additives designed to enhance immune function have been developed, including nucleotide products, beta-glucans, prebiotics and probiotics. These products are not yet widely used in rainbow trout farming. One of the main factors that affect the health and growth of the fish is feeding. Remember that, in spite of the huge variety of dry food, fish eating a natural food, growing and multiplying better than other. Starlet - a predator feeds on benthic invertebrates, insects and their larvae. In general, the main diet consists of crustaceans, larvae of caddis flies and chironomid larvae. Granulated feed the most effective in the industrial cultivation of sturgeon in particular, starlet that determines the relevance of their improvement with the use of new components with high protein content, porncake or minerals [66].

1.4 Zeolites are natural minerals used in veterinary science Zeolites are crystalline solids structures made of silicon, aluminum and oxygen that form a framework with cavities and channels inside where cations, water and/or small molecules may reside. They are often also referred to as molecular sieves. Many of them occur naturally as minerals, and are extensively mined in many parts of the world finding applications in industry and medicine. However, most of zeolites have been made synthetically some of them made for commercial use while others created by scientists to study their chemistry. At present, there are 191 unique zeolite frameworks identified [68], and over 40 naturally occurring zeolite framework are known. Zeolites are crystalline aluminosilicates with open 3D framework structures built of SiO4 and AlO4 tetrahedra linked to each other by sharing all the oxygen atoms to form regular intra-crystalline cavities and channels of molecular dimensions. A defining feature of zeolites is that their frameworks are made up of 4-coordinated atoms forming tetrahedra. These tetrahedra are linked together by their corners and make a rich variety of beautiful structures. The framework structure may contain linked cages, cavities or channels, which are big enough to

23 allow small molecules to enter. The system of large voids explains the consistent low specific density of these compounds. In zeolites used for various applications, the voids are interconnected and form long wide channels of various sizes depending on the compound. These channels allow the easy drift of the resident ions and molecules into and out of the structure. The aluminosilicate framework is negatively charged and attracts the positive cations that reside in cages to compensate negative charge of the framework. Unlike most other tectosilicates [69], zeolites have largeer cages in their structures. The naturally occurring zeolites are an important group of minerals for industrial and other purposes. The discovery in 1957 of largedeposits of relatively high purity zeolite minerals in volcanic tuffs in the western United States and in a number of other countries represents the beginning of the commercial natural zeolite era. Prior to that time there was no recognized indication that zeolite minerals with properties useful as molecular sieve materials occurred in large deposits. Commercialization of the natural zeolites chabazite, erionite, and mordenite as molecular sieve zeolites commenced in 1962 with their introduction as new adsorbent materials with improved stability characteristics. The applications of clinoptiolite in radioactive waste recovery and in waste water treatment during the same period of the 60's were based not only on superior stability characteristics but also high cation exchange selectivity for cesium, strontium, and for ammonium ion. The well known and industrially important zeolites have been discovered in 1950-1970 and may be classified into three groups according to Al/Si ratio in their frameworks [70]. The properties of the porous materials depend both on the pore structures and the chemistry of the framework. The continuously increasing demands for materials with highly specific chemical and physical properties as zeolites have inspired scientists to make new porous materials with unique structures [71]. Zeolites are widely distributed in nature, and animals can easily find their outputs on the ground and eat in large quantities. These places are called "licks". Zeolites are regularly found in the feces of deer, mountain goats, deer, bears, birds, etc. Zeolites - minerals from the group of water aluminosilicates of alkali and alkaline earth elements, volcanic-sedimentary origin with a tetrahedral structural framework comprising cavities (voids), occupied by cations and water molecules. Currently there are more than 40 types of structural natural zeolites, the most common of which are clinoptilolite, heulandite, Phillips, Lomond, mordenite, erionite, chabazite, ferrierite, analcime [72-73]. In 1756, F. Cronstedt discovered swelling (increase in volume of the sample, accompanied by release of water) stilbite (mineral family of hydrated aluminum silicates ) when heated. So he coined the term "zeolite" (in Greek " boiling stone " - " ZEO " - boils "Litos" - stone). It turned out that a similar property is possessed and other minerals of this family: clinoptilolite, mordenite, chabazite, etc., which

24 can be represented as a crystalline aluminosilicate anion whose charge is compensated by cations of sodium, potassium, calcium or magnesium. Unlike crystalline hydrates also emit significant quantities of water by heating, zeolites do not absorb and emit only water but also various other molecules without changing the crystalline structure. Besides zeolites absorption unlike coordination bonding in crystalline due to the phenomenon of adsorption - concentrating substances from the gas phase onto a solid surface (adsorbent) or within the volume formed by its pore structure. Common to the entire zeolite group of minerals is the presence of three- dimensional alyumokremnekislo-native skeleton forming a system of cavities and channels, which are located in the alkaline, alkaline cations and water molecules. The cations and water molecules are loosely bound to Caracas and may be partially or completely substituted (removed) by ion exchange and the dehydration, and reversible without destruction of the zeolite framework. Stripped water zeolite is a microporous crystalline "sponge" pore volume, which makes up 50 % of the zeolite skeleton. This "sponge", which has a mouth diameter of 0.3 to 1 nm (depending on the kind of zeolite), is a highly adsorbent. Diameter inlets "sponge" is strictly defined dimensions. In connection with this, there is a so-called molecular sieve adsorption of molecules at the selection of the gas in the liquid. Properties of zeolites allow separate molecular mixtures even in cases where the difference in the molecular size of 10-20 pm. Ion-exchange properties of zeolites are determined on the chemical affinity of ions in the crystal structure of zeolite. Thus, as well as during adsorption of the molecules must comply with the dimensions of the inlet openings in the zeolite framework and the substituent ions. Ion exchange on zeolites unable to allocate ions which extraction another method often is of great complexity. Ionositovy effect of allowing adsorbed gas and liquid systems, the nitrogen vapor, CO2, SO2, H2S, Cl2, NH3. Zeolite is a natural mineral, which is formed by a crystal lattice of silicon atoms, aluminum and oxygen. Excess negative charge on the oxygen atoms allows them to bind a large number of different metals: alkali, alkaline earth and heavy. They can be interchanged. This determines the ion-exchange properties of zeolites. In addition, zeolites have a microporous structure. The total area of the interior surface is enormous: 1 g mineral corresponds to 800 m2. This determines the molecular sieve properties of zeolites, which allows them to connect different substances are not ionic nature (ammonia, urea, and even low molecular weight proteins). Adsorbing the protein on the compound it and they can exhibit catalytic properties. Adsorption sites in zeolites are hydroxyl groups, various cations and Lewis centers [74-76]. Natural zeolites are a valuable form of unconventional materials. There are about 30 species: clinoptilolite, mordenite, chabazite, erionite, Phillips neyrolit, laumontite, etc. Its unique Cato and catalytic properties, they are increasingly used in various technologies.

25

Adding natural zeolite of the clinoptilolite type to feed mixtures in low doses of about 1–2% has influences on very important functions heretofore not recorded by other natural compounds [77]. In Slovak Republic the presence of mycotoxins in feed mixtures of both plant and animal origin has been observed. The most abundant types of mycotoxins include the aflatoxins B1, B2, G1, G2, M1 and ochratoxin A. They are produced by the toxinogenic phyla Aspergillus flavus and Aspergillus parasiticus. Depending on weather conditions in the given year a 40– 80% contamination of the cereal grain by fungi of the Fusarium genus, namely Fusarium graminearum and Fusarium poae. An effective tool for degradation of the toxins in animal feed was found in the zeolite of the clinoptilolite type from Nizˇny´ Hrabovec in the concentration of 0.9–1.7 kg/100 kg of feed. The effect was demonstrated in the improved condition of the animals and weight increases. In general, in monogastric animals it was found that salts of organic acids were effective as feed additives; this was true especially of calcium formate (HCOO)2Ca [78] which has bactericidal and acidifying effects in the feed mixture while at the same time influencing the pH adjustment of the digestive tract and increasing the activity of the enzyme pepsin. Finally, this leads to increased growth intensity in animals and better feed conversion. To support these positive effects half of the calcium formate was replaced by zeolite from the Nizny Hrabovec deposit which at the same time played the role of carrier for formic acid (HCOOH). In the course of animal digestion process the formate is transformed to formic acid and only in this form can influence the digestion process. The feed additive containing 50% calcium formate, 8% formic acid and 42% of natural zeolite of the clinoptilolite type proved more effective at increasing the growth intensity and improving feed conversion in pigs and poultry [79]. Following are some of the test results for the above feed additive (developed by CHEMKO Strazske under the trade name of ZEOFORM containing 42% of natural zeolite of the clinoptilolite type) [80]. The feed mixture contained 1.5% of the feed additive. After the first month of feed administering the animals in the test group showed a weight increase of 13.4 kg as compared to 8.3 kg in the control group. The daily increase was 0.47 kg in the test group against 0.29 kg in the control group. At the same time the consumption of feed per kg of weight increase was 3.13 kg in the test group and 4.7 kg in the control group. Natural clinoptilolite type zeolite is a zeolite minerals most commonly used in the world. Currently, it is widely used in many fields of industrial technology [81- 84], agricultural production [82], and ecology [83] as well as in other fields such as medicine [84]. Clinoptilolites widely distributed in nature. Zeolite - porous natural mineral containing up to 70% clinoptilolite, and as impurities - montmorillonite, quartz, feldspar, opal, volcanic glass, etc. Clinoptilolite is a high-silica zeolite with silica to alumina ratio from 3.5 to 10.5 and contains an average of 60 % silica. Do clinoptilolite diameter input windows in the cavity is 0.4 nm. Clinoptilolite has strictly calibrated pore size (approximately 4 angstroms). From this it follows that it is able to show the sorption properties only with respect

26 to ions of macro- and micronutrients and compounds with small size (methane, hydrogen sulfide, ammonia, etc.), not engaging in direct interaction with vitamins, amino acids, proteins and other complex organic compounds. Natural zeolite in the gastrointestinal tract is not absorbed into the blood stream is not himself as crystal, and transits , interacting only at the level of selective ion exchange and selective sorption in contact with blood and lymphatic vessels of the intestinal wall, giving or taking the micro- macro catalyzing biochemical reactions. The largest volume of studies on the effectiveness of natural zeolites held in livestock, poultry, fish farming, fur farming, animal health, and plant. Comprehensive studies have been carried out, ranging from the study of the quality of zeolitic tuffs and ending biomedical evaluation of animal products, ensuring environmental cleanliness and safety of milk, meat, eggs, fish and other raw materials and products. When this has been installed throughout the high efficiency of zeolites, and return one tenge acquisition costs and their use in scientific experiments, and in terms of production is from 5 to 15 tenge. Feasibility study on the use of zeolites in assessing the effectiveness of livestock and poultry have been launched by the Institute of Experimental Veterinary of Siberia and the Far East (Shadrin A.M.) in 1979. In the future, to work with zeolites in agriculture were connected to many other research, educational, and industrial enterprises. The reason for the increased interest in zeolites in agriculture served as its following properties: excrete heavy metals, reduce activity in the intestine aktinomitsenov and some antibiotics, adhere various microorganisms, without affecting the pathogenic E. coli, immobilized enzymes of the gastrointestinal tract, thus enhancing their activity and stability, to regulate the water and salt conditions in the intestine: regulates the content of free water than promotes formation of denser stool, is an additional source macro ultra micro minerals, slowing advancement of the food bolus in the gastro-intestinal tract, mainly in the small intestine. Action zeolites on animals are divided into primary and secondary. The primary mechanism of action of zeolites on the animal organism are determined: localized in the cavities and channels framework cations sodium, potassium, calcium, magnesium and other changes in the ionic composition of the chyme of the stomach and intestines, pH normalization and optimization of environmental conditions for the work of digestive enzymes; catalytic properties of zeolites performance impact on the assimilation of proteins, fats, carbohydrates, prolonging the period of biological activity of enzymes, as well as increased proteolytic and amylolytic activity of the intestine (change in the activity of enzyme systems of digestion, changing the suction capability of the gastrointestinal mucosa); adsorption properties: the adsorption of ammonium ions, indole, skatole, mercaptan or other endotoxins that in first, reduces their content in the blood flowing from the intestine into - second causes numerous mediated changes in other systems and organs (liver, lymph, blood, etc.). (High adsorption activity of natural zeolites has a positive effect in dyspepsia, reduces fermentation increases the synthesis of bacterial protein and calcium silicate).

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A secondary mechanism of action on the organism of animal’s zeolites leads to: tissue respiration change, as evidenced by the increase in hemoglobin in blood, a significant increase in red blood cells in many cases, modification of protein metabolism and energy homeostasis. Reflection of these processes is the growth of total protein, free amino acids of the majority of the plasma, additional deposits in the liver glycogen, increased activity of catalase and blood aminotransferase, increased levels of vitamins A, B, C and carotene in the blood, liver, eggs; modulation of the specific and nonspecific resistance: an increase in the level of albumin and globulins in blood alkaline reserve, an increase in titer of agglutinins and a sharp increase in the phagocytic activity of leukocytes indicate increased resistance of the organism ; relative increase in muscle tissue. It is known that the growth of muscle tissue controlled by the thymus, so relative increase muscle mass indirectly indicates the participation of the central immune organs in the implementation of zeolites influence on the body. The consequence of this is to increase the participation of stability of experimental animals to infectious diseases, is actually observed; reduce mortality of birds, the disappearance of negative behaviors, improve the appearance of the experimental facilities, increase the strength of the egg shell, their fertility and the percentage yield of chicks from eggs of hens experienced indicates an increase in the viability of the animals; activation of enzyme systems, improving the protein composition of the blood, reduced levels of ammonia in it. Feed efficiency with the addition of zeolite is increased significantly due to the adsorption of toxins and antimetabolites zeolites present in the feed. Sorption properties of the zeolites creates conditions for the removal of toxins and anti-metabolites, ions, metal-containing ions which do not contain metals, heterocyclic compounds, toxins, bacteria and mold, and pesticide residues etc. Change of protein, fat and carbohydrate metabolism when fed to animals zeolite zeolite gives reason to believe to be potentially effective state control of the internal environment and use products for the control and prevention of adverse complications of transition states in animals, as well as to develop methods to improve the productivity of their exposure to the factors of industrial technology content animals. Through a series of studies have examined the use of zeolites in veterinary medicine and animal husbandry. Work on the use of zeolites in veterinary practice and animal husbandry is very numerous. One of the typical works on this subject [85] is to study the influence of impurities introduced into the diet of cows (zeolite and humolita) on mineral and protein metabolism, the process of calving. Found that feeding cows in the winter and summer- grazing period for 60 days before calving and 45 days after calving zeolite and humolita at 0.5 liters per 1 kg of body weight does not affect the performance of the physiological status of cows. At the same time - increasing the specific gravity of crude bone, enhances the content of calcium and phosphorus, 5- 40 % by 2-11 % in the bone tissue and increase the calcium-phosphorus ratio in

28 winter from 1,14:1 to 1 in the control, 36:1 in case of using zeolite and 1,40:1 - humolita and high content of total protein in the blood by 5-13 %. In this paper, N.I. Yarovan [86] the possibility of using natural zeolites Hotynetskom to prevent the development of oxidative stress and correction of disturbances in the antioxidant system in cows in the treatment of diseases of the reproductive system and in unfavorable conditions for keeping and feeding winter - stall period. Considered absolutely proven that zeolites sorb heavy metals [87-98], free radicals [99-102], the decomposition products and toxins [103-114] radioactive elements [115-120], thereby taking on a significant portion of antitoxic function of the body , especially the liver. In this paper, D.A. Zasyekin [121] has shown that the oral administration to laboratory animals, salts of heavy metals (Cu, Zn, Pb, Cd, Sr) their level increases in parenchymal organs of rats of 2 to 365 times . In this laboratory animals subcompensated developing metabolic acidosis, inhibited the activity of enzymes, hyperglycemia develops, increasing ureogenez violated protidosynthetic liver function, reduced concentration and changes ratio of keto and glucogenic, essential and nonessential amino acids. To eliminate heavy metals from the organs and tissues of laboratory animals tested the effectiveness of natural and synthetic sorbents (zeolite, saponite, palygorskite, humolit, polisorb-M envet-1). Were established their positive effect. These sorbents, contributing to the removal of excess heavy metals to the parameters of the MPC, do not cause changes in clinical parameters, normalize metabolism of proteins, carbohydrates, lipids, minerals in the body, which indicates the possibility of their wide use as detoxicants and preventive medicines. In [122-123] the absorbent activity of some zeolites for glucose that can be used in the treatment of diabetes. In this paper, N.G. Kuramshina et al. [124], in particular, assessing the impact on the chickens and zeolites Sibaiskiy Baimak fields. Zeolite was added at a concentration of 3% by weight of the feed. In the experimental group compared to the control increased significantly the number of erythrocytes, leukocytes and hemoglobin. In egg yolks in the experimental group compared to the control increased amounts of carotene, vitamin A and B. In this paper, S.N. Zedgenizovoy and O.V. Prosekin [125] also gives positive results of experiments with the addition of zeolite in feed for chickens. In particular, the addition of the zeolite at a concentration of 5 % by weight of the observed increase in the total feed amount of the erythrocytes and leukocytes and the increase of hemoglobin, respectively. It should be noted also that the zeolite in the form of a 5% granulated feed supplement increased the live weight of the birds without causing any adverse effect on the general condition and the blood of laying hens. In this paper, S.G. Lumbunova et al. [126] showed that the zeolites with a particle size of 0.5-2.5 mm were good mineral supplements for adult birds. Adding to feed hens Sotnikovsk poultry farm in an amount of 3.6 % of the weight of the

29 economy provided the equivalent amount of feed, increased the safety of livestock by 1.5-2.0 %, egg production increased by 5-8%, improved the quality of the egg shell. Furthermore, the use of the zeolite had a noticeable deodorizing effect. In general, similar results contained in the works of V.N. Khaustova [127] and A.M. Shadrina et al. [128-129]. Regarding the use of zeolites in the fisheries sector appeared the following article: [130-136]. In one of these studies comprehensively studied patterns of accumulation of nitrate and nitrite in water and fish products, proposed ways to reduce their toxicity by using zeolites and preparations based on them. Developed and implemented in the fishery complex treatment and preventive measures, including animal health requirements for growing fish to contaminants, recommendations for diagnosis, treatment and prevention of poisoning fish. First work on testing in fish zeolites showed the possibility of their successful application in the form of feed additives [137-139]. There is reason to hope that the use of zeolites and other aluminosilicate zeolite series geylendit - feeding fish can give a significant positive effect. Adding natural zeolite clinoptilolite in feed at low doses of about 1-2 % impact on a very important function Adding clinoptilolite in feed has exactly the same effect as the use of antibiotics. Previously performed studies in this area can be characterized as fragmented and not giving enough information to substantiate the use of zeolites in tuffaceous composition of fish feed [140].

1.5 Chemical composition and nutritional value of fish Fish is a food of excellent nutritional value, providing high quality protein and a wide variety of vitamins and minerals, including vitamins A and D, phosphorus, magnesium, selenium, and iodine in marine fish. Its protein - like that of meat - is easily digestible and favourably complements dietary protein provided by cereals and legumes that are typically consumed in many developing countries. Experts agree that, even in small quantities, fish can have a significant positive impact in improving the quality of dietary protein by complementing the essential amino acids that are often present in low quantities in vegetable-based diets. But recent research shows that fish is much more than just an alternative source of animal protein. Fish oils in fatty fish are the richest source of a type of fat that is vital to normal brain development in unborn babies and infants. Without adequate amounts of these fatty acids, normal brain development does not take place [141]. Fish is one of the most important sources of animal protein available, and has been widely accepted as a good source of protein and other elements for the maintenance of a healthy body [142]. The protein content of fish varies from 15-24% [143]. The proteins in fish muscle tissue can be divided into three groups:  structural proteins (actin, myosin, tropormyosin and actomyosin) which constitute 70-80 percent of the total protein content (compared with 40

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percent in mammals). These proteins are soluble in neutral salt solutions of fairly high ionic strength (0,5 M);  sarcoplasmic proteins (myoalbumin, globulin and enzymes) which are soluble in neutral salt solutions of low ionic strength (<0,15 M). This fraction constitutes 25-30 percent of the protein;  connective tissue proteins (collagen), which constitute approximately 3 percent of the protein in teleostei and about 10 percent in elasmobranchii (compared with 17 percent in mammals) Structural proteins make up the contractile apparatus responsible for muscle movement. The amino-acid composition is roughly similar to corresponding proteins in mammalian muscle, although the physical properties can differ slightly. When the proteins are denatured under controlled conditions, their properties may be utilized for technological purposes. A good example is the production of surimi- based products in which the gel-forming ability of the myofibrillar proteins is used. After salt and stabilizers are added to a washed, minced preparation of muscle proteins and after a controlled heating and cooling procedure, the proteins form a very strong gel [144]. The majority of sarcoplasmic proteins are enzymes participating in cell metabolism, such as the anaerobic energy conversion from glycogen to ATP. If the organelles within the muscle cells are broken, this protein fraction may also contain the metabolic enzymes localized inside the endoplasmatic reticulum, mitochondria and lysosomes. The fact that the composition of the sarcoplasmic protein fraction changes when the organelles are broken was suggested as a method for differentiating fresh from frozen fish, under the assumption that the organelles were intact until freezing. However, it was later stated that these methods should be used with great caution as some of the enzymes are liberated from the organelles during iced storage of fish as well. The proteins in the sarcoplasmic fraction are excellently suited to distinguishing fish species, as each species has a characteristic band pattern when separated by the isoelectric focusing method. The chemical and physical properties of collagen proteins are different in tissues such as skin, swim bladder and the myocommata in muscle. In general, collagen fibrils form a delicate network structure with varying complexity in the different connective tissues in a pattern similar to that found in mammals. However, the collagen in fish is much more thermolabile and contains fewer, but more labile, cross-links than collagen from warm-blooded vertebrates. Different fish species contain varying amounts of collagen in body tissues. This has led to a theory that the distribution of collagen may reflect the swimming behaviour of the species. Furthermore, the varying amounts and varying types of collagen in different fishes may also have an influence on the textural properties of fish muscle [145]. Amino acids play a central role as the building blocks of proteins and as intermediates in metabolism and further help to maintain health and vitality. There

31 are 20 amino acids that can be found in the human body, 18 of which are important in human nutrition. Eight amino acids cannot be synthesised de novo by humans and other mammals and hence must be supplied in the diet; therefore they are called essential amino acids [146]. The essential amino acids are lysine, methionine, threonine, tryptophan, isoleucine, leucine, phenylalanine and valine. Failure to obtain enough of even one of the essential amino acids results in the degradation of the muscle proteins in the body. Moreover, there is a group of amino acids which are not normally required in the diet but which must be exogenously supplied to specific populations under special conditions, such as intensive growth, stress, or in some disease states. Such amino acids have been classified as semi-essential. This group includes histidine, serine and arginine. The remaining amino acids (alanine, cystine, glycine, aspartic acid, glutamic acid, proline and tyrosine) are synthesised by the organism in sufficient amounts and hence are classified as nonessential amino acids [147]. The amino acid composition of fish protein is similar to that of other animal proteins. All of the essential amino acids are present. The lysine content is high, making fish a good supplement to cereal protein. The lysine content may be about 30% greater than that found in beef and about eight times that in bread. The histidine level is also high, especially in the red meat fish. Bacterial decarboxylation of histidine and the subsequent formation of histamine in scombroid fishes may create food poisoning problems. Tryptophan and methionine are characteristically low, with methionine (or cysteine) being the limiting amino acid. However, cysteine is still for times more abundant in fish protein than in casein. The three general classes of proteins found in fish are the sarcoplasmatic proteins, the myofibrillar proteins, and the connective tissue proteins. Sarcoplasmic protein or myogen makes up 20 to 30% of the total protein content. This fraction is composed chiefly of enzymes, most of which resemble those found in mammalian sarcoplasm. Two enzymes which are unique to fish are thiaminase and anserinase. The remaining sarcoplasmic fraction, about 0,5%, consists of the colored hemocyanin proteins and cytochrome c. These are usually present in low concentrations, especially in the white-fleshed fish [148-150]. All fish apparently require the same ten indispensable or essential dietary amino acids required by most other animals. These amino acids are: arginine (arg), histidine (his), isoleucine (ile), leucine (leu), lysine (lys), methionine (met), phenylalanine (phe), threonine (thr), tryptophan (trp) and valine (val). Cystine spares part of methionine requirement in rainbow trout. Although not proved, tyrosine may spare phenylalanine in fish diets [151]. Recent studies by Ketola give quantitative data on the dietary requirements of rainbow and lake trout for lysine and arginine. Signs of deficiency of dietary amino acids in fishes generally include reduced growth, poor feed conversion and reduced appetite. A few amino acid deficiencies lead to anatomical abnormalities. For instance, deficiency of methionine causes lake trout to develop bilateral lens cataracts and suffer poor growth and survival.

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This effect of methionine deficiency on cataracts was confirmed in rainbow trout. Tryptophan deficiency causes scoliosis and lordosis in sockeye salmon and rainbow trout, but apparently not in the calfish. Further effects of tryptophan deficiency in rainbow trout include abnormal calcium deposits in the kidney and the bony plates surrounding the notochord and sheath. Lysine deficiency in rainbow trout causes fin rot, i.e. loss of much of the fin, but not in lake trout [152]. Studies conducted at the Tunison Laboratory demonstrate that fish egg amino acid amino acid pattern serves as a useful guide for formulating feeds and studying amino acid requirements for rainbow trout and Atlantic salmon. For instance, a study by Rumsey and Ketola showed the various approaches to appraising the deficient or limiting amino acids in commercial soybean meal (49% crude protein) as a sole source of protein in diets of rainbow trout [153]. Lipids are the generic names assigned to a group of fat soluble compounds found in the tissues of plants and animals,: and are broadly classified as: a) fats, b) phospholipids, c) sphingomyelins, d) waxes, and e) sterols. Fats are the fatty acid esters of glycerol and are the primary energy depots of animals. These are used for long-term energy requirements during periods of extensive exercise or during periods of inadequate food and energy intake. Fish have the unique capability of metabolizing these compounds readily and, as a result, can exist for long periods of time under conditions of food deprivation. A typical example is the many weeks of migration by salmon in their return upstream to spawn; stored lipid deposits are burned for fuel to enable body processes to continue during the strenuous journey [154]. The lipid content of fish varies depending on the type of fish, the time of year and what the fish feeds on [155-156]. Meat of fish contains insignificant amounts of carbohydrates in the form of glycogen and high percentage of water (60–86%) [157]. The content of vitamins and minerals in meat of freshwater fish is very favourable [158]. The energy value of fish meat is directly proportional to fat content. It was found that fish fats vary greatly in regard to the percentage of saturated and unsaturated fatty acids and usually contain 15–36% saturated fatty acids [159-161] and 58–85% unsaturated fatty acids [162-163]. The most important unsaturated fatty acids are linoleic and linolenic acid, which are essential and should be ingested in the body by food. Results referring to meat quality of starlet are different in communications by various authors, with differences mostly caused due to the analysis of fish of different age, breeding systems and food and because of that, there are wide ranges of fat content in carp, from 2,3 to 16,8%, while varying slightly less in case of protein and protein content in range from 14 to 18% [164-166]. Beside polyunsaturated fatty acids, fish fats contain cholesterol. Fish meat contains similar amount of cholesterol (49–92 mg/100 g) as pork or beef (45–84 mg/100 g) and cholesterol content is not correlated with fat content [167]. Dietary linolenic acid or ethyl linolenate (18:3 w 3) gives a positive growth response for rainbow trout which may be attributed to a dietary requirement for w 3 fatty acids.

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Rainbow trout, a cold water fish, requires 3 fatty acids as EFA in the diet. The EFA requirement can be met by 1 percent 18:3 3 in the diet. Inclusion of 18:2 6 in the diet may result in some improvement in growth and feed conversion compared to EFA deficient diets; however, the 6 fatty acids will not prevent some EFA deficiency symptoms such as the "shock syndrome". Although it is clear that rainbow trout require 3 fatty acids, it remains to be shown conclusively whether some dietary level of 6 fatty acid is essential. In all the above studies with rainbow trout, dietary 18:2 6 or 18:3 3 were readily converted to C-20 and C-22 PUFA of the same series, and 18:3 3 or 22:6 3 had similar EFA value for rainbow trout. Either 20:5 3 or 22:6 3 is superior to 18:3 3 in an EFA value for rainbow trout, and the former two fatty acids in combination are superior to either alone. This is consistent with data for mammals, where 20:4 6 has higher EFA value than 18:2 6. The superior nutritional value of C-20 and C-22 carbon 3-PUFA is further supported by the excellent growth promoting effects of dietary fish oils such as pollock liver oil and salmon oil for rainbow trout [167- 174]. The amount of vitamins and minerals is species-specific and can furthermore vary with season. In general, fish meat is a good source of the B vitamins and, in the case of fatty species, also of the A and D vitamins. Some freshwater species such as carp have high thiaminase actitity so the thiamine content in these species is usually low. As for minerals, fish meat is regarded as a valuable source of calcium and phosphorus in particular but also of iron, copper and selenium. Saltwater fish have a high content of iodine. The vitamin content is comparable to that of mammals except in the case of the A and D vitamins which are found in large amounts in the meat of fatty species and in abundance in the liver of species such as cod and halibut. It should be noted that the sodium content of fish meat is relatively low which makes it suitable for low-sodium diets. In aquacultured fish, the contents of vitamins and minerals are considered to reflect the composition of the corresponding components in the fish feed, although the observed data should be interpreted with great caution [175]. In order to protect the n-3 polyunsaturated fatty acids, considered of great importance both for fish and human health, vitamin E may be added to the fish feed as an antioxidant. It has been shown that the resulting level of vitamin E in the fish tissue corresponds to the concentration in the feed [176]. Seafood is also an excellent source of minerals. Fish are one of the most important sources of calcium. The soft bones of small fish such as sardines and smelts and canned varieties like salmon are especially valuable sources of calcium. Other minerals in seafood include Zinc (oysters and crustaceans), Iron (oysters, bluefish, and shrimp), Copper (oysters, crabs, and lobster), Potassium (mussels, scallops, and clams), Iodine, Phosphorus, and Selenium. The study of mineral elements present in living organisms is of biological importance; since many of such elements take part in some metabolic processes and are known to be indispensable to all living things. The body usually contains

34 small amount of these minerals, some of which are essential nutrients, been components of many enzymes system and metabolic mechanisms, and as such contribute to the growth of the fish. The most important mineral salts are that of calcium, sodium, potassium, phosphorous, iron, chlorine while many others are also needed in trace amounts. The deficiency in these principal nutritional mineral elements induces a lot of malfunctioning; as it reduces productivity and causes diseases, such as inability of blood to clot, osteoporosis, anemia etc [177].

1.6 Pesticide residues in feed and fish Nowadays, more than 800 different kinds of pesticides are used for the control of insects, rodents, fungi and unwanted plants in the process of agricultural production. Although most of them leave the products or degrade in soil, water and atmosphere, some trace amounts of pesticide residues can be transferred to humans via the food chain, being potentially harmful to human health [178]. Pest control in intensive agriculture involves treatment of crops (fruits, vegetables, cereals, etc) pre and post harvest stages, rodenticides are employed in the post-harvest storage stage, and fungicides are applied at any stage of the process depending on the crop. These chemicals can be transferred from plant to animal via the food chain. Furthermore, breeding animals and their accommodation can themselves be sprayed with pesticide solution to prevent pest infestations. Consequently, both these contamination routes can lead to bioaccumulation of persistent pesticides in food products of animal origin such as meat, fat, fish, eggs and milk [179-180]. During the last decades much attention has been given to this group of substances and the international level after it became apparent that they are transported through the environment and critical concentrations have been reached in some areas even in places where they have never been produced or used. Several countries banned the use of Organochlorine Pesticides (OCPs) during the 1970s and 1980s, although many of them continue to been used by other countries. OCPs have been identified as one of the major classes of environmental contaminants because of their persistence, long-range transport ability and human and animal toxic effects. OCPs are carcinogenic in animals as well as in human. The immunotoxicity of selected OCPs has been also documented in vitro [181], in vivo [182], as well as in animals, in human fetal, neonatal and infant immune systems [183-187]. A growing number of epidemiological studies have investigated blood or adipose levels of OCPs and their metabolites in relation with cancer, neurodevelopmental effects, immuno‐toxicity and reproductive efficiency [188- 191]. The main sources of OCPs in the human diet are foods of animal origin and environmental exposure. It has been concluded that humans are exposed to toxic compounds via diet in a much higher degree compared to other exposure routes such as inhalation and dermal exposure. Low volatility and high stability, together with lipophilic behaviour, are responsible critical factor for their persistence in the environment (air, water and soil) and subsequent concentration in fatty tissues through the food chain. Therefore, it’s important to identify and to monitor levels

35 of OCPs in foodstuff of animal origin (meat and tissues that contain fat, milk and dairy products, eggs, honey and fish). The main pathway for the OCPs contamination of animal food is the ingestion of the contaminated food and/or water by the animals [191-193]. Breeding animals can accumulate persistent organic pollutants from contaminated feed and water, and/or from pesticides application in livestock areas (treatment of cowshed, pigsties, sheepfold etc.) [194- 197]. The use of feedstuffs in farms has become indispensable for animal diet in developed countries because of increasingly higher production requirements. Animal feed plays an important part in the food chain and has implication for the composition and quality of the livestock products that people consume. Therefore, the control of OCPs residues in animal feed is mandatory as well as the control in fatty tissues. Organochlorine pesticides (OCPs) were intensively used in agriculture to protect cultivated plants in mid-twentieth century. 1,1,1-Trichloro-2,2-bis(4 chlorophenyl)ethane (DDT), one of the common OCPs, was used to prevent spreading of malaria and other vector-borne diseases such as dengue, leishmaniasis and Japanese encephalitis through the prevention of growth of mosquito [198-200]. After OCPs were used widely in soil and plants for some years and due to their relative stability and bioaccumulation property, these persistent chemicals can be transferred and magnified to higher trophic level through the food chain. Consequently, OCP residues are present in fatty foods, both foods of animal origin such as meat, eggs and milk, and of plant origin such as vegetable oil, nuts, oat and olives. Besides, these chemicals are widely distributed in the environment, which provides another route of unwanted intake in human. Nevertheless, human exposure occurs still primarily via low level food contamination. Since their mode of action is by targeting system or enzymes in the pest which may be identical or very similar to system or enzymes in human beings, these OCPs pose risks to human health and the environment. Thus, monitoring of OCPs residues in food becomes a routine analysis of pesticides monitoring laboratories. All US government pesticides datasets showed that persistent OCP residues were surprisingly common in certain foods despite being off the market for over 30 years. Residues of dieldrin, in particular, posed substantial risks in certain root crops. About one quarter of samples of organically labelled fresh produce contained pesticides residues, compared with about three quarters of conventional samples. Among the contaminated organic vegetable samples, about 60% of them were contaminated with OCPs. After some OCPs were banned for use since the 80s, common daily food items such as eggs, milk, poultry, meat and fish have been used for monitoring the residuals levels of OCPs. As regards food of animal origin, one efficient way to avoid large-scale contamination is to control and monitor the levels of OCPs residues present in animal feeds before being fed to the husbandry animals [201-204]. At the same time, public health safety authorities should constantly monitor the OCPs in animal food commodities as the major source of human background exposure to OCPs is through food of animal origin. Most persistent organic

36 pollutant (POPs) are OCPs, namely, aldrin, endrin, chlordane, DDT and hexachlorobenzene (HCB). They have been banned for agricultural or domestic use in Europe, North America and many countries of South America, in accordance with Stockholm Convention in 1980s. However, some OCPs are still used, e.g. DDT is used to control the growth of mosquito that spread malaria or as antifouling agent in some developing countries [205-207]. Residues of OCPs have been detected in breast milk (including DDT, HCB and HCH isomers) in contaminated areas. Recently, the scope of POPs was extended to include nine plus one chemicals. Among these new POPs, chlordecone, lindane, α-HCH, β-HCH, pentachlorobenzene (PeCB) and endosulfan, also belong to OCPs. In order to fulfil the requirements of the Stockholm convention, the participating countries have to develop their own implementation plant to monitor the background level and collate the exposure data. To ensure the pesticide residues are not found in food of feed at levels presenting an unacceptable risk for human consumption, maximum residue levels (MRLs) have therefore been set by the European Commission [208-212]. MRLs are the upper legal concentration limits for pesticides in or on food or feed. They are set for a wide range of food commodities of plant and animal origin, and they usually apply to the product as placed in the market. MRLs are not simply set as toxicological threshold levels; they are derived after a comprehensive assessment of the properties of the active substance and the residues behaviour on treated crops. Both the periodic estimation of human exposure to persistent organic pollutants and the establishment by the EU authorities of MRLs in foods have required the development of analytical methods suitable for research purposes and inspection programmes.

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2 OWN RESEARCHES 2.1 Materials and methods 2.1.1 Research materials The experimental part of the work was carried out in between 2011 - 2014 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 JSC "Kazakh Academy of Nutrition" (Almaty), the Kazakh Scientific Research Institute of Oncology and Radiology (Almaty) and the Radioecological research center (Semey), in the branch of the Republican Veterinary Laboratory (Almaty). The physical and mechanical properties and mineral composition of Tseofish unconventional feed additive were determined. The zeolite used in the experiments was sourced from the Chankanay plant producing zeolitic materials. The mineral composition of the feed additive Tseofish (natural zeolite) was determined at the Kazakh-Japan Center with a scanning electron microscope SEM JEOL JSM 6430F. The subject of the research were fingerlings and yearlings of rainbow trout and sterlet (Oncorhynchus mykiss, and Acipenser ruthenus), which were given pelleted with added natural zeolite-clinoptilolite (which contained a higer concentration of the active substance - clinoptilolite); Chankanay zeolite contains between 65-75%, so this zeolite does not require enrichment. Clinoptilolite tuff from this source had a high content of the zeolite (55.6%) with impurities including quartz and feldspar. The chemical composition of the zeolite (Table 1) was characterized by the sodium-calcium ionic form. The arsenic content was 0.002%, fluorine - 0.003% lead - 0.0015%, which did not exceed the norms imposed on the zeolites used in feed production. The zeolite in the feed composition was administered shredded fine corresponding sieve bandwidth №067. According to the solution of specific problems the research scheme sought optimal solutions (Table 1). Zeolite (clinoptilolite) as a feed additive tested in the composition of the feed – RGM-2M and RGM-9PO (in two versions), intended for fingerlings and yearlings of rainbow trout and sterlet fingerlings. Experimental studies were conducted in the Republican State Enterprise "Kapshagay spawning and nursery industry", the Committee for Fisheries, Ministry of Agriculture of the Republic of Kazakhstan, in Enbekshikazakh district of Almaty region. For this purpose five groups of rainbow trout and five groups of starlet were selected, which were kept in specialized pools, rectangular shape. The optimal water temperature for starlets was 20-26°C while for rainbow trout - 15-18°C, the optimum concentration of oxygen dissolved in water for starlet was 20-21°C, the optimum concentration of dissolved oxygen in the water for trout was 9-12 mg/L. (MARK-302 E - dissolved oxygen analyzer).

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Feed prescription RGM-2M was offered: for fingerlings: fish meal - 4.6%, meat and bone - 9%, the blood - 5%, wheat - 11% algae - 1%, hay - 2%, skim dry - 9%, hydrolytic yeast – 4%, soybean meal (flour) - 6% sunflower meal (flour) - 2% fish oil (vegetable oil) - 4% premix - 1%. Fish were fed 6-8 times a day. For feeding yearlings a dough-like mixture of this composition was used, comprising: beef spleen – 50%, meat and bone meal – 10%, flour from the pupae of silkworm – 5%, fishmeal – 5%, flour from grain wastages – 12%, phosphatides- 10%, yeast – 5%, fish fat – 1%, salt and a premix – 1%. For sterlets, as the base feed sturgeon pellets RGM - 9PO were used. In feed for fish of the experimental group were added 1%, 2%, 3% and 4% of zeolite per 1 kg of feed. The duration of feeding the experimental fish was 90 days. Fish in the control group received the basic diet of fish farms in the experimental group, with 1 x 90-th day of cultivation, were added to the feed of 1%, 2%, 3%, 4% by weight of the zeolite feed. To study the effect of the feed additive Tseofish on meat production and organoleptic characteristics of meat, indicators of the chemical composition of the meat were collected from 2 groups of young trout and starlets of 20 individuals with a mean weight of 15 g.

Figure 1 - Arrangement of the experiment for feeding the experimental fish (rainbow trout and sterlet) with granulated feed, using the feed additive Tseofish

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Table 1 Scheme of the study

Experimental substantiation of application of Tseofish in aquaculture

stage I Study of the effect Tseofish on the fish body

1. Development of dosages and additive regime of Tseofish

The scheme of BD+1% BD + 2% BD + 3% BD+4% feed additive BD Tseofish Tseofish Tseofish Tseofish application

(days) Experimental groups, n = 300 Control I scheme (1 - group 1 group 3 group 5 group 7 group 9 61) (trout) II scheme (1- 61) (sterlet) group 2 group 4 group 6 group 8 group 10

Footnote: 1 - BD – basic diet

1. Study the chemical composition of the Tseofish feed additive; 2. Analysis of meat productivity and sensory characteristics of fish meat. 3. The study of the chemical composition of fish meat when used in feed NFA Tseofish; 4. Study of the amino acid composition of fish meat when using NFA Tseofish; 5. Determination of fatty acid composition of muscle tissue of fish with the addition of Tseofish ; 6. Analysis of the mineral composition of fish meat when used in feed NFA Tseofish; 7. Study of the vitamin content of fish meat when used in feed NFA Tseofish.

stage II Study the effect of Tseofish on morphological changes of organs and tissues

stage III

Study the effect of Tseofish on haematological indices

IVstage Study the effect of Tseofish on the elimination pesticides in meat and fish40 feeds

1. Study the effect of Tseofish on the elimination of pesticides in the meat of fish 2. Study the effect of Tseofish on elimination of pesticides in fish feeds

2.1.2 Research methods Methods of organoleptic, chemical, amino acid, fatty acid, vitamin, mineral analysis were examined: young rainbow trout and staret are selected on the research of fish farms and ponds where there has been fertilizing fish feed additive Tseofish. 2.1.2.1 Organoleptic methods Sampling and organoleptic tests were carried out according to the VG "Veterinary-sanitary examination of products of animal origin," ST RK 1802-2008 Fish, seafood and their products. Acceptance rules and sampling were carried out by GOST 7631-85 "Fish, marine mammals, marine invertebrates and derived products. Acceptance rules, organoleptic methods of quality assessment, sampling methods of laboratory tests, "ST RK 1803-2008 Fish. Methods of sensory evaluation. Fish were gutted and packed in ice and transported to the laboratory on the day of slaughter, all analyzes were performed on the day following sampling. Organs analyzed: liver, kidneys, intestines cut in the laboratory. Quality of fish meat studied by organoleptic methods GOST 7631-85, and the determined of the appearance, examining the skin and fins, the state of mucus, its quantity and quality, especially the scales, eyes, mouth, color of the gills and internal organs and muscles: their consistency, odour, and clear broth. Colour, shape, pattern and extent of mucilaginized gill structure of the petals were noted. Exterior view was determined by the fatness of fish, regarding its outer covering, slime, gills and abdomen. Consistency of the fish meat was evaluated by palpation of the fleshy parts. The smell of the fish was determined in the anal area, gills, and superficial mucus. The smell evaluation procedure was as follows: clean knife were poked into the body of fish, removed and the smell was immediately determined. Punctures were made in different places of the body: in the muscle between the dorsal fin and the head, in the site of injury and mechanical damage, and from the interior through the anus. The knife should be administered with caution to avoid unnecessary damage to the fish. The smell of fish was determined as the sample pulping. The taste was determined without cooking, checked by assaying thin slices of cut from the fleshy part of the fish. Opening of fish was with the use of scissors. Two cuts were made: one on the white line - from the anus to the gill arches and the second from the same point on the white line to the head. The left half of the abdominal wall was removed and the intestines, liver, pancreas, spleen and kidneys

41 were examined. As for the internal organs, the freshness of the fish was evaluated. After extracting the internal organs the peritoneum and the presence or absence of the red stripe along the spine were assessed. 2.1.2.2 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. Prior to analysis, fish fillets were ground together with the skin. 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 10 min. Hot broth was filtered through a pad of 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 2 ml portion of the filtrate was poured into a vial to 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 5 min, 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 sulphide a 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 inner surface of the neck of the flask and 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 fish, the liquid is immediately coloured a bluish color (subsequently turning to brown). If the meat is of doubtable freshness and substandard, bluish color does not appear. For determining the amino ammoniacal nitrogen, 20 g of an average sample of minced meat is added to 100 ml of distilled water and pressed for 15 minutes, shaking every 5 min. The infusion was then filtered through filter paper. Next, of 100 ml 10 ml 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 42 flask was neutralized with 0.1N solution of potassium hydroxide which had a slightly pink colour. In the neutralized extract 10 ml of 40% formalin solution was added, as previously neutralized to a phenolphthalein until of a slightly 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 10 ml of the filtrate meat extract in milligrams was calculated by multiplying the flow of 0.1N. potassium hydroxide titration in the second by a factor of 1.4. If necessary, adjustments were made for the alkali titer. The content of amino-ammonia nitrogen in 10 ml of extract from fresh meat does not exceed 1.26 mg, in a meat of suspicious freshness - from 1.27 to 1.69 mg, 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 the intensity of its colour compared, on the white background compared, with the colour of liquids on the bichromate scale. Assessment about the freshness of fish: fresh fish up to 1.0 on the Nessler scale; of suspicious freshness - 1,2-1,4; stale - 1.6 and above. 2.1.2.3 Methods for determining of chemical composition of fish The chemical composition of fish meat was determined: moisture by drying at 105°C, Soxhlet fat, total protein - a modified method of Kjeldahl, minerals - incineration in a muffle furnace. Ash was determined using a muffle furnace by heating at 550°C for eight hours. The caloric content of the meat was determined by the formula suggested by Alexandrov. 2.1.2.4 Method for determining the amino acid composition of fish The amino acid composition of the fish meat was determined by ion exchange chromatography with an automatic amino acid analyzer AAA 881 "Czech". Prior to hydrolysis the samples were degreased with petroleum ether at room temperature. Acid hydrolysis (6 N HCL) was carried out at 110°C for 24 hours for all amino acids except methionine, and other sulfur-containing amino acids. Methionine was measured after performic acid oxidation followed by acid hydrolysis (AOAC, 1980). For calibration the standard Beckman amino acid solution was used. All assays were performed in a double repetition. Assessment of amines was calculated according to the recommendations of the FAO/WHO (1973). 2.1.2.5 Methods of determining fatty acid composition of fish Fatty acid composition of the fish meat was determined by gas-liquid chromatography Agilent. Fatty acids of fish samples were determined as fatty acid methyl esters (FAMEs). Before analysis the head, tail, fins and entrails of fish were removed. Edible tissue, fillet with skin was homogenized. Fish samples were prepared using direct saponification of KOH/methanol, followed by derivatization (trimethylsilyl)

43 diazomethane method (Aldai et al. 2006). Composition of FAs analyzed by a GC Clarus 500 autosampler (Perkin Elmer, USA) equipped with a flame ionization detector and a fused silica capillary Supelco-SP-2330 fused silica capillary column (30 m, internal diameter 0.25 mm, 0.20 mm thickness polyethylene film) (Bellefonte, PA). The oven temperature was 140°C, held for 5 minutes, then raised to 200°C at a rate of 4°C min-1 and then to 220°C at a rate of 1°C min-1. The injector and detector temperatures were set at 220°C and 280°C respectively. The sample size was 1 mm and the carrier gas was maintained at 16 psi. Cleavage used was 1: 100. Separate FAMEs (fatty acid methyl esters) were determined in accordance with the same retention time peaks using standard mixture component Supelco 37 FAME Mix. All data were subjected to unilateral analysis of variance (ANOVA) using Statistica 8,0 computer program to test the effects of the experimental diets. Multi- band test Ducane and critical ranges were used to test differences between means. Differences were considered significant at P <0,05. All results are expressed as mean ± SD. 2.1.2.6 Method for determining the mineral composition of fish The mineral composition of fish meat was assessed as follows: iron was determined by the colorimetric method using GOST 26928-86; while for the determination of calcium and magnesium GOST-09-066-02 was used. For determining mineral content the method of dry ashing was used. That is took place in an ashing muffle furnace at 400-600°C. In order to accelerate the process of loosening using NH (CO) a sample was placed in a bomb chamber filled with oxygen and closed. The combustion process took three minutes. The resultant ash containing metal oxides was treated with a solution of HCl (1: 1) to obtain the soluble metal chlorides. 2.1.2.7 Method for determining the vitamin composition of fish 2.1.2.7.1 Equipment Separation and quantification were performed on a Hewlett Packard Series 1100 system. The HPLC system used in this study consisted of pump (HP 1100 series Binary Pump), a sample injector (model 7725/7725i), an UV detector (HP 1100 series Diode Array Detector) and a 20 µl loop. The laboratory was equipped with a water bath (Memert WB-14, Germany) and laboratory oven (Venticell 111, BMT Medical Technology s.r.o., Czech Republic). 2.1.2.7.2 Reagents and solutions All solutions were prepared with analytical reagent grade compounds. Vitamins were provided by Supelco. Pyridoxine was obtained from Sigma-Aldrich. The enzyme used in the sample preparation Clara- diastase was from Fluka. Methanol (purity: 99.8%) was obtained from Merck KGa (Darmstadt, Germany). KH2PO4 was obtained Merck KGa (Darmstadt, Germany). Deionized water was purified by Aqua osmotic system (Aquaosmotic, Tisnov, Czech Republic). 2.1.2.7.3 Samples and reference materials Calibration curve and quantification. The calibration curves were obtained by plotting different concentration (μg.ml-1) against peak area. All the standard

44 stock solutions were prepared by dissolving 20.0 mg appropriate vitamin in 100 ml mobile phase to obtain a concentration of 20.0 µg.ml-1. The working solutions were prepared by suitable dilutions of the stock solutions with the mobile phase. For each vitamin, a series of six concentration points (1.0, 2.0, 3.0, 4.0 5.0 and 6.0 µg.ml-1) were injected into the column under the same chromatographic conditions. The regression equations were obtained from calibration curve of each standard vitamin and used for the calculation of quantify of B3, B5 and B6 in sample. 2.1.2.7.4 Sample preparation The extraction of vitamins by enzymatic mixture has been recommended (Barna et al., 1994, Arella et al., 1996, Ndaw et al., 2000) for hydrolysis of the sample prior to its quantification. The extraction procedure used in our study was based on a combination of acid and enzymatic hydrolysis. The esters and protein bounded vitamins are converted to clear vitamin forms by the using of this process. A 5g homogenized portion of meat were suspended in 50 ml of 0.1 mol.dm- 3 HCI and then autoclaved at 1210C for 30 min. Upon cooling, the pH was adjusted to 4.0 with 2 mol.dm-3 sodium acetate and 2.0 ml of 2% Clara-diastase suspension was added to the sample. Then samples were submitted to enzymatic digestion for 18 h at 370C. After cooling the sample was made up to 100 ml with 0.01 mol.dm-3 HCl and filtered through paper filter (Filtrak, No390). The extracts were stored in refrigerator at 40C until analysis. Before injection into the column samples were filtered through 0.45 µm pore size nylon filter. Sample preparation procedures were done under dim light and then kept in the dark vials. 2.1.2.7.5 Chromatographic conditions The samples were separated on the reverse-phase Supelcosil LC 8 column (4,6 x 150 mm, 5µm). The injection volume was 20 µl and the temperature of column was thermostated at 250C. The mobile phase was 0.1 mol.dm-3 KH2PO4 (pH 7.0)-methanol, 90:10. The flow rate was 1.0 ml.min-1 and time of analysis was 30 min. The detector’s wavelengths were set at 220 nm and 204 nm for B1, B3 and B6 respectively. Calibration curves were set like dependence peak area on concentration and were used for quantitative analysis. 2.1.2.8. Methods of histological examination of meat Histological examination was carried out in accordance with GOST 19496-93 "Meat. The method of histological examination, "GOST R 51604-2000" Meat and meat products. Histological method for identifying the composition. "Sampling of fish meat was carried out in accordance with GOST 19496 and GOST 23481. 2.1.2.8.1 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 overnight. To the filtered protein was added glycerol at a ratio of 2:1, stirred and 0.1 g of camphor was added to prevent rotting. The resultant mixture was applied

45 to a degreased glass slides triturated using a gauze swab, and dried over a flame burner. 2.1.2.8.2 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. 2.1.2.8.3 Preparation of Ehrlich hematoxylin Ehrlich's haematoxylin was prepared by mixing 20 cm3 of a 10% alcoholic solution of, 80 cm3 of 96% alcohol, 100 cm3 of glycerol, 100 cm3 of distilled water, 10 cm3 of glacial acetic acid and 3 g of potassium alum. The resulting solution was poured into a wide-mouth jar, 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 aqueous solution 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 3 cm3, 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 hours in 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 15x15x4 mm 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 fine brush 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 a dissecting needle. Then the slice covered with dry filter paper and, by pressing the paper by hand, stuck onto a glass slide. 2.1.2.8.4 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 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. Thereafter, slices were placed under a coverslip. Prepared histological preparations were examined under a biological microscope BIOLAM M-1. Cooked preparations were examined and photographed using a microscope «LEICADM 4000 B». 2.1.2.9 Hematological study of fish

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Blood was taken from hungry fish in well-seasoned aerated water for 10 minutes after capture. Blood was collected from the tail artery. For blood drawing a syringe with an injection needle was used. Scales for on-site collection of blood were removed with a scalpel, the slimy skin was wiped and disinfected with 70% alcohol. Blood was collected in a Pasteur pipette, transferred to a watch glass and the necessary amount for hematological studies was collected. The haemoglobin content was determined by the method of Sali. For this purpose, a graduated pipette gemometra Sali to the mark "2" is used, and eyedropper poured a decinormal solution of hydrochloric acid. In a capillary pipette from gemometra Sali the blood was introduced up to the 20 mcl and a solution of hydrochloric acid was added. The resulting mixture was stirred with a glass rod and left for 10 minutes. After this time into the tube distilled water was poured dropwise and stirred with a glass rod. The test solution was colour-selected to match the colour of the liquid in the standard tubes. The amount of haemoglobin was counted on the lower meniscus of the working solution in the graduated tube (figures in grams % expressed in g/l), 1 g of 10% g/l. The number of erythrocytes was determined by test-tube in the Goryaev chamber. A 4 ml Hendriks solution was poured into the chemical test-tube by graduated pipette. The blood was added from gemometr Sali into the capillary pipette to the 20 mcl mark and added into the tube, gently washing the capillary several times. Cover glass to grind in chamber Goryaev before the appearance of Newton rings. The tube contents were thoroughly mixed and using a Pasteur pipette filled a Goryaev chamber. After 1-2 minutes, the numbers of erythrocytes under the microscope in 80 small squares arranged diagonally were counted. To determine the number of erythrocytes in 1 mm it is sufficient to multiply the number obtained by counting the number of erythrocytes per 10,000. The number of leukocytes was determined by the direct method of counting the number of them in a Goryaev chamber. Blood from a pipette typed into the mixer for counting of erythrocytes of mammals up to the 0,5 or 1 mark. The tip of the mixer was wiped with cotton, collected into a mixer with solution A (25 mg neytralrot and 0-6 g of sodium chloride dissolved in 100 ml distilled water) to half expansion mixer 101 was then filled to the mark with Solution B (12 mg of crystal violet, 3.8 mg of sodium citrate and 0.4 ml of formalin were dissolved in 100 ml of distilled water). After filling from the mixer the rubber tube was removed by pinching it between thumb and middle finger, the blood was stirred with the dilution fluid. The mixer was left in horizontal position for 10 minutes and then stirred again. The first 2-3 drops that came out of the mixer were ignored, and only the subsequent drops were introduced into the counting chamber. A coverslip was previously coupled onto a counting chamber before the appearance of Newton's rings. Under the action of the solutions A and B there is a vital staining of the blood cells. The nuclei of leukocytes are stained a dark purple-red colour, while the protoplasm is stained pink. Erythrocytes are only weakly stained. Thus are erythrocytes and

47 leukocytes distinguished in the chamber. The number of leukocytes were counted in 80 large squares, and their number was determined by the formula: x = mx250xU / n, Where: X - number of cells in 1 mm2, M - is the total number of cells in counted squares U - the degree of dilution of the blood, n - number of scanned squares

2.1.2.10 Determination of pesticide residues in feed and fish meat 2.1.2.10.1 Samples and reagents In this study 50 samples of feed were investigated, including 25 samples RGM-2M, 25 samples RGM-9PO. The research material consists of samples from two areas of Kazakhstan, as presented in Fig. 1. All samples have been obtained from local farmers according to ISO 24333:2009 (ISO, 2009). Pesticide-free cereal samples were used as blanks to spike for the validation process. All reagents used were of analytical grade. Acetone, acetonitrile, n-hexane, and methanol for pesticide residue analysis were provided by J.T. Baker (Deventer, Holland). Florisil (60–100 mesh) (J.T. Baker, Deventer, Holland), anhydrous sodium sulfate (Fluka, Seelze-Hannover, Germany) and silica gel (Merck, Darmstadt, Germany) were activated at 600 °C. 2.1.2.10.2 Standarts Pesticides were obtained from the Dr. Ehrenstorfer Laboratory (Germany) and are listed in Table 1. Pesticide standard stock solutions (purity for all standards >95%) of various concentrations were prepared in acetone and stored at 4°C. Standard working solutions were prepared by dissolving 1 ml of stock solution with a mixture hexane/acetone (9:1; v/v). 2.1.2.10.3 Sample preparation A representative portion of feed was blended. 2.0 g of a sample was put in a mortar with 4.0 g Florisil and was manually mixed using a pestle to produce a homogeneous mixture (4 min). The mixed material was transferred to a glass column (1.5 cm i.d. 30 cm length) containing anhydrous sodium sulfate (2.5 g) and silica gel (2.5 g), and an additional layer of anhydrous sodium sulfate (2.5 g) was placed on the bottom of the column. The analytes were eluted using 25 ml of an acetone/methanol mixture (9:1). The extract was evaporated at a temperature of about 40 °C and then diluted in 2 ml of hexane/acetone (9:1, v/v). A 1.7 ml of extract was placed in an SPEC-18 column, which was rinsed twice with 5 ml acetonitrile earlier and not allowed to dry. Analytes were eluted with 15 ml acetonitrile. The extract was collected in a round-bottomed flask and the solvent was distilled off in an evaporator until dry (bath temperature 40°C). The remainder was dissolved in 1.7 ml of a hexane–acetone mixture (9:1, v/v). The final solution was put into a GC vessel and placed on the rack of the autosampler. The scheme of feed sample preparation is shown in Fig. 2. 2.1.2.10.4 Instrumentation and chromatographic conditions

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GC analysis was performed with an Agilent (Waldbronn, Germany) model 7890 A gas chromatograph equipped with ECD and NPD with HP-5 column ((5% -Phenyl)- methylpolysiloxane; 30 m -0.32 mm and film thickness 0.50 lm) and Chemstation chromatography manager data acquisition and processing system (Hewlett–Packard, version A.10.2). For confirmation of residues a mid-polarity column: HP-35 ((35%-Phenyl)-methylpolysiloxane; 30 m 0.32 mm and film thickness 0.50 lm) was used. The operating conditions were as follows: for detectors an injector temperature of 210°C; carrier gas: helium at a flow-rate of 3.0 ml min-1; detector temperature: 300°C (ECD and NPD); gas: nitrogen at a flow- rate of 57 ml min-1 (ECD) and 8 ml min-1 (NPD), hydrogen 3.0 ml min-1, air 60 ml min-1; for oven – initial temperature: 100°C increase at a rate of 10°C/min up to 250°C and held for 25 min, from 250 to 300°C at a rate of 50°C/min and held for 5 min at the final temperature (ECD and NPD). The volume of final sample extract was injected at 210°C in splitless mode (purge off time 2 min) was 2 ll injected and the peak height was compared to that of the calibration standards (in matrices) to determine the residue quantitatively. 2.1.2.10.5 Method of validation In this study wheat samples were selected as a representative commodity for the validation of the method in determination of pesticide residue (Sanco, 2011). Calibration curves were obtained from matrix-matching calibration solutions. The lowest concentration level in the calibration curve was established as a limit of detection. Calibration standards were prepared by the addition of 1 ml spiking solutions to a blank matrix of the wheat to produce a final concentration of 1st range 0.001– 0.05 mg kg-1, 2nd range 0.1–0.5 mg kg-1 and 3rd range 0.5–2.5 mg kg-1. Recovery data were obtained at three range concentrations in the matrix, each day using blank cereal samples (wheat) in accordance with European Commission (EC) guidelines (Sanco, 2011). Blank samples (2.0 g) after homogenization were spiked by addition of appropriate volumes of pesticide standard mixture in solution hexane/acetone (9:1, v/v) and were left for 1 h (equilibration times) and then prepared according to the procedure described above. Method accuracy and precision were evaluated by performing recovery studies. The precision was expressed as the relative standard deviation (RSD). Accuracy can be measured by analyzing samples with known concentration and comparing the measured values with the true values. The limit of quantification (LOQ) was defined as the lowest concentration of the analyte that could be quantified with acceptable precision and accuracy. The limit of detection (LOD) was defined as the lowest concentration of the analyte in a sample which could be detected but not necessarily quantified. The LOQ and LOD were evaluated as the signal-to-noise ratios (S/N) of 10:1 and 3:1 for the pesticide, respectively. As a part of external quality control, laboratory has been successfully participating in six proficiency tests within European Commission’s Proficiency Testing Program (EUPT). The participation in EC tests is mandatory for all official laboratories undertaking analysis of these commodities within the framework of official inspections of pesticide residues. Additionally, the comparisons between laboratories and the national reference laboratory (Plant

49

Protection Institute – National Research Institute, Poznan) have been performed. The method has been accredited according to ISO/IEC 17025 by the Polish Centre for Accreditation with the accreditation number AB839. 2.1.2.11. Statistical processing of results Statistical processing of the material (the mean values and confidence intervals) was performed using the statistical functions of the Microsoft Excel program. The significance of differences in the values of each sample was determined by Student's t-test criterion for the program.

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3 RESEARCH RESULTS

3.1 Physico-chemical properties of non-traditional feed additive Tseofish

Nontraditional feed additive Tseofish - a natural mineral zeolite Chankanay field, which upon treatment at the plant had been pulverized into small particles. The raw material is available in Kazakhstan zeolite tuff sedimentary origin, containing 60-80% of the zeolite-clinoptilolite and montmorillonite as impurities, quartz, feldspar, opal and others. Zeolites - a large group of similar composition and properties of minerals, water and sodium aluminum silicates of calcium from the subclass framework silicates, glass or pearlescent, known for their ability to give and re-absorb water, depending on temperature and humidity. Another important property of zeolites is the cation exchange capacity - they are able to selectively isolate and re-absorb a variety of substances, as well as to exchange cations. Zeolites (aluminosilicates with crystalline structure) got its name from the Greek words "OSS" and "lithos", which means "effervescing stones." When heated zeolites swell and release water, which is always present in them due to the unusual structure (rocks penetrated by strictly oriented internal pore-tubules). The main property of zeolites is the ability to actively neutralize various geopathic radiations. Scientists established that the "food minerals" have a significant impact on the rate of growth of the animals, as well as on the understanding of basic food components: proteins, fats and carbohydrates. Many of these minerals have been used successfully for decades in breeding cattle, pigs, poultry and fish in the United States, Japan, Germany, Hungary and Bulgaria. The most common representatives of the group of zeolites are natrolite, chabazite, heulandite, stilbite (desmin), mordenite, thomsonit, laumontite, clinoptilolite. The most widespread species of zeolite called clinoptilolite, as it has a unique absorption and healing properties. The name "clinoptilolite" comes from the Greek words "wedge" - tilted (Tilt and microcrystalline individuals), "ptilon" - "down" and "lithos" - "stone." White, light gray, beige, pink.

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Figure 2 - Shredded zeolite

Figure 3 - The zeolites in powder form

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In the sensory evaluation of alternative feed additive Tseofish pay attention to the appearance, color, odor, and taste, solubility in water and acid (H2SO4), fineness of grinding on the natural mineral. In appearance alternative feed additive Tseofish is a fine crystalline powder light color with pinkish tinge, odorless, solids with a crystal size of less than 0,05 mm. This feed additive has a taste of chalk or clay. This feed additive is not in water, but easily dissolved in sulfuric acid (H2SO4). Organoleptic characteristics of the non-traditional feed additive Tseofish are shown in Table 2. Zeolite characterized by high water capacity, water resistance (96-98%), absorptive capacity. Technological properties such as flowability, dispersibility, traceability not go beyond acceptable parameters specific for this group of natural minerals.

Table 2 - Organoleptic characteristics of the feed additive Tseofish Name of parameters Parameter Exterior view fine chopped powder mass Solubility in water insoluble acid (H2SO4) easily soluble Color white with a pink tinge Taste tastes of chalk Odor odorless Size, the residue on the sieve with holes of 10 diameter 5mm,%, not more

The chemical composition of zeolitic raw material is one of the important indicators of quality. From the Si / Al ratio and cation composition of zeolites vary their ion exchange properties and resistance to aggressive media and high temperatures and the ability to modification, and a number of other technological characteristics. High silica zeolite: clinoptilolite, mordenite, and others. - The most resistant to the above factors, as well as ion exchangers are good and are widely used in adsorption technology, for this reason they are referred to commercially valuable minerals. Zeolites from different deposits by chemical composition somewhat different from each other, so we investigated the performance of the chemical composition of the zeolite experienced (Table 3).

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Table 3 - Chemical composition of nontraditional feed additive Tseofish

Components wt.% NFA Tseofish SiO2 57,90 Al2O3 16,40 TiO2 0,14 Fe2O3 5,45 FeO 0,61 CaO 5,12 MgO 2,32 MnO 0,10 Na2O 2,03 K2O 2,56 P2O5 0,13

For a sample characterized by a high content of SiO2 (about 56%) and the Si / Al ≥5, indicating the presence of a high silica zeolite (clinoptilolite). In zeolites Chankanay deposits, compared with zeolites other fields, there is a decrease and increase of the contents of K Ca and Na. Thus, the cationic composition of zeolites Chankanay origin can be attributed to a calcium-sodium. In zeolites Chankanay field markedly increased content of manganese, magnesium, phosphorus, aluminum. Electron microscopic studies revealed that zeolites generally have complicated relief microsurface formed microcrystals and units represented in the majority of cases the fine mass. Aggregates of microcrystals are concentrated in mikrozheodah and microcracks located in the breed are relatively evenly. In preparing the produced natural zeolite is generally crushed to a particle size of 5-20 mm by using the crusher. Subsequently, this material is pulverized using an ultrasonic dezintregratora to obtain a fraction with a particle size of from 1-2 to 10 microns.

Figure 4 - Electronic photo of clinoptilolite crystals. Magnification × 500

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Comparison of the results of mechanical and ultrasonic method of grinding material as the main process of preparation of the powder is illustrated by photographs obtained with a scanning electron microscope (Figure 3,4). At the bottom of the field images are the options of shooting modes (from left to right: power supply voltage, magnification, image scale, job number and the current time). In Figure 6, we can see the result of mechanical grinding zeolites for 30 minutes. Here smaller, lighter particles on the background of the zeolite crystals and presented montmorillonite. As can be seen from the photograph (magnification - 500) dispersion of reduced particle size zeolite and from 20 to 50 m, while on the zeolite particles are clearly visible sharp edges. Figure 3 shows the result of mechanical grinding of zeolites for 1 hours. The magnification - 500.

3.2 Toxicological evaluation of non-traditional feed additive Tseofish

To date, the acute toxicity when given enterally zeolites used for feeding purposes is not revealed. Harmlessness, and on the other hand, the biological effectiveness of different types of feeding aluminosilicates shows a number of research experiments conducted over the past 10-20 years by various researchers []. In the study of the toxicology of natural mineral clinoptilolite was unable to determine the value of a lethal dose for animals, birds and fish. Enteral administration of maximum doses administered to experimental animals, birds, fish in spicy experiment do not cause functional changes in the body []. Insufficient knowledge of the matter on the toxicity of natural mineral deposits in Kazakhstan served as the basis for conducting experiments. Toxicity study of nontraditional feed additive Tseofish (zeolite-based) on the body of fish, guppies showed that the use of this feed additive in the composition of fish feed toxic effects are not observed in the experimental groups of fish guppy mortality was observed. Signs of intoxication of an organism of fish were found. Furthermore, no abnormalities were detected in the behavior, in a general state and appetite. Guppy fish were mobile and active, well-eaten feed, kept all reflexes. Based on the studies found that the components of the feed additive in the tested concentrations did not cause pronounced toxicity and can be attributed to the group of non-toxic drugs.

Table 4 - Evaluation of the toxicity of non-traditional feed additive Tseofish on guppies fish The The number of dead guppies, exe. degree of 1 2 3 4 The toxicity experimental experimental experiment experimental control group group al group group group Non-toxic 15 15 15 15 15 Slightly - - - - - toxic

55

Toxic - - - - - In the table 4 shows the results of evaluation of the toxicity of non- traditional feed additive Tseofish on guppies’ fish. According to studies the toxicity of the feed additive was nontoxic. The fish were healthy, active, well-eaten food. Consequently, most likely the fish died due to lack of food and not due to toxicity of the feed additive Tseofish. Thus, the results of the study it can be concluded that nontraditional feed additive Tseofish is not toxic at a ratio of 1-4% of the fish, as they are made of completely harmless natural mineral zeolite. This is most likely due to a lack of food, and has no relation to the toxicity of the feed additive.

3.3 Study of the effect of non-traditional feed additive Tseofish at fish- biological and hematological parameters of the body of fish

3.3.1 Fish breeding and biological indicators of valuable fish species when using nontraditional feed additive Tseofish

The aim of research was the fish breeding and biological and physiological substantiation of application of non-traditional feed additive Tseofish in the composition of feed for rainbow trout and sterlet on the example of yearlings. The goal identified the need to address the following objectives: to study the effect of natural zeolite on growth, development and physiological state of yearlings rainbow trout and sterlet, when introduced into their diet feed additive Tseofish. The experiments were conducted in accordance with the scheme of studies presented in Table 5.

Table 5 - Scheme of the experiment Group № Number of fish Conditions Control 50 100% B.D. 1 experimental 50 99% B.D.+ 1% NFA Tseofish 2 experimental 50 98% B.D. + 2% NFA Tseofish 3 experimental 50 97% B.D. + 3% NFA Tseofish 4 experimental 50 96% B.D. + 4% NFA Tseofish Note: Abbreviation: BD - The basic diet

Conditions, the hydrological and water temperature regimes were the same for all the experimental and control groups of rainbow trout and sterlet. The objects of study were rainbow trout and sterlet. In the experimental and control groups of rainbow trout initial mass of fish at landing was in the experimental groups, respectively 341g, 348g, 343g, 345g, the control - 346g,

56 while the sterlet initial mass 266g, 264g, 266g, 269g, 267g. Experimental and control groups of rainbow trout were grown in pools with size 2x2x0,7 m with once-through water supply. The water temperature in the pools at the beginning of the experiments was 11.5°C and the end of the experiments increased to 19.9°C. This temperature is above the optimum temperature for growth of rainbow trout, in connection with which experience has been discontinued. Duration of the experiment was 61 days (from 21.05 to 20.07.2012). The average temperature for the entire period of experiment was 16.9 °C. The oxygen content – 5.5-7.2 mg/l, pH – 6.6-7.2. The next stage of research after the experiment on the fish to feed additives for yearlings rainbow trout has been the study of their impact on all fish processes of vital activity. The first were evaluated fish breeding and biological indicators of growing trout two yearlings, and then studies have been conducted to assess the physiological state of the fish. The results of studies on the effectiveness of the use of NFA Tseofish as the additive to mixed fodder for growing fingerlings of rainbow trout are shown below in Table 6. The final weight of trout yearlings in all experimental variants was higher in comparison with the results of the cultivation of fish in the control groups.

Table 6 - Results of growing of rainbow trout yearlings in the diet was added NFA Tseofish The average mass, g Growth Group the initial final Absolute The The growth, relative average g growth,% daily growth,% 1 experimental 341±19,6 457±21,63 116 34,01 0,47 (1 % Tseofish) 2 experimental 348±19,9 453±19,5 105 30,17 0,42 (2% Tseofish) 3 experimental 343±13,4 461±15,3 118 34,1 0,48 (3% Tseofish) 4 experimental 345±17,6 469±16,2 124 35,9 0,49 (4% Tseofish) Control 346±20,1 428±17,9 82 23,6 0,40

During the research it was found that the introduction of feed NFA Tseofish had a positive effect on the growth of rainbow trout juvenile and its physiological state. Absolute growth of juvenile up to 61 days of growing in the experimental pools with addition NFA Tseofish in an amount of 1%, 2%, 3%, 4% relative to the control was equal to respectively 116g, 105g, 118g, 124g, while in the control group its figure was 82g. 57

Indicators of average daily gain in the first variant (1% Tseofish) exceeded those of the control group and second test group at 0,07 and 0,05%. The index daily average gain of fish reached a maximum level (0.49%) in the fourth test group. Survival in all variants was 100%.

Table 7 - Results of growing sterlet two yearlings in the diet was added NFA Tseofish Average mass, g Growth Group initial final The The The absolute relative average growth, growth, daily g % growth,%

1 experimental 266 ±16,1 380±15,7 114 42,8 0,57 (1 % Tseofish) 2 experimental 264±26,3 383±16,9 119 45,07 0,60 (2% Tseofish) 3 experimental 266±19,4 388±14,6 122 45,8 0,61 (3% Tseofish) 4 experimental 269±20,7 393±18,8 124 46,09 0,61 (4% Tseofish) Control 267±14,9 353±19,0 86 32,2 0,45

In comparative evaluation of fish breeding and biological indicators sterlet yearlings with two-year rainbow trout in which feed was added NFA Tseofish at different doses apparent significant difference in absolute weight gain of the fish, that is, if juvenile rainbow trout his figure was in the range 82-116 g, then in the young sterlet he is 86-124 g (Table 7). And there was the percentage increase in the relative weight growth sterlet on average by 10,8%. Perhaps this is due to the fact that the sterlet was set the correct temperature. It should be noted that the starlet relates to heat-loving fish and their feeding and growth occur most rapidly at 18-25 °C. Thus, in the course of the research, it was found that the introduction of the feed additive Tseofish in fish feed had a positive effect on the growth of rainbow trout and sterlet juvenile and their physiological state. Absolute growth of juveniles for 61 day of growing in the experimental basins with the addition of NFA Tseofish in quantities of 1, 2, 3, 4% in relatiion to the control was higher. Indicators of average daily growth of fish in experimental groups also exceeded the control group in the two species of fish.

3.3.2 Hematologic characteristics of valuable fish species when using a feed additive Tseofish

58

The degree of influence of NFA Tseofish on the physiological properties of rainbow trout can be characterized by such indicators as the composition of the blood. Mixed fodder including in its composition NFA Tseofish, had no significant effect on the physiological state of the fish. Table 8 shows the data of hematological parameters in the blood of rainbow trout when used in the composition of feed the feed additive Tseofish. The content of hemoglobin in the blood of rainbow trout yearlings in the experimental variants was 93,5 g/L (1% NFA Tseofish), 94,1 g / l (2% NFA Tseofish), 95,7 g / L (3% NFA Tseofish), 96,3 g/L (4% NFA Tseofish), respectively, in the control group this indicator was – 87,6 g/L (the differences are significant at p ≤ 0,05.). As seen from Table 8, in the experimental and control groups, the differences were small. According to the results obtained hematological data can be judged about good physiological condition of growing fish. The differences were not found between the other hematological parameters - the number of white blood cells, as well as the number of immature red blood cells. All of them are included in the scope of the normative values. At a rate of 50- 60 thousand/mm3 level of leucocytes of fish in experimental groups was 58,6x109/l (1 experimental group), 59,3x109/l (2 experimental group), 59,7x109/l (3 experimental group), and 60,6x109/l (4 experimental group) against 58,3x109/l for fish in the control group. The bulk of the white blood cells were represented by lymphocytes (90,32%), which testify to absence in the body of rainbow trout deviations from the physiological norm. We observed an increase of monocytes total number in the experimental group, if the number of monocytes of rainbow trout was equal to 5,0% at the contol group, already at the 4 experimental group the monocytes total number was 5,6%, which is relatively high. The total protein content in the blood serum increased gradually in the experimental groups of fish, which feed was added NFA Tseofish at different doses. The maximum value of its indicator reached in the 4 experimental group – 7,12%. It indicates that the feed additive Tseofish has no negative effect on the total protein content in serum. Results of the study of hematological parameters when used as in the composition of fish feed the feed additive Tseofish are shown in the Table 9. According to the study of hematological parameters of sterlet blood in experimental groups, which was added to the feed NFA Tseofish found that the feed additive does not cause significant changes in the composition of blood, all blood parameters were within normal limits. The hemoglobin content in the blood of the sterlet experimental groups (50,6-54,3 g/L) compared with the control group (49.9 g/L) were higher, but their performance is not exceeded physiological norm for that species of fish. Studies of sterlet blood parameters which received feed without NFA Tseofish and with its introduction in the feed showed that during the period when the feed was added NFA Tseofish, erythrocyte concentration and total protein in blood serum of sterlet yearlings were slightly higher (respectively, p <0, 05 and p <0,001) than in the period when in feed was not added this feed additive.

59

Increasing the concentration of total protein in serum (p <0.05), most likely due to an increase in growth indicators of farmed fish. In peripheral blood of examined sterlet during consumption of the feed additive Tseofish except mature erythrocytes forms mentioned young forms of erythrocytes - polychromatic and oxyphilic normoblasts, which was associated with activation of erythropoiesis in this period, with activation of metabolic processes in the body the examined fish. In the blood of sterlet experimental groups their proportion ranged from 0,591 to 0,621x1012/l. Content of leukocytes, lymphocytes and neutrophils also remained in normal. Monocytes also remained within the normal range, significant deviations in the experienced period were not observed. Thus, the physiological state of the two-year-rainbow trout and sterlet, when added to the composition of feed NFA Tseofish in quantities of 1, 2, 3, and 4% at the end of cultivation in line with the norm, as in the experimental groups and the control. From this we can conclude that the feed additive does not cause abnormalities in the physiological status of the fish and it can be used as feed additives in the feed.

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Table 8 - Effect of non-traditional feed additive Tseofish on hematological indicators of rainbow trout meat (n = 20) The experimental groups 1 2 3 4 Unit of Control group Physiological norm Indicators experimental experimental experimental experimental measurement (BD) for rainbow trout group (1% group (2% group (3% group (4% Tseofish) Tseofish) Tseofish) Tseofish) Hemoglobin g/l 87,6±1,3 93,5±0,39 94,1±0,16 95,7±0,13 96,3±0,36 70-110 Erythrocytes х1012/l 1,13 ±0,06 1,15±0,31 1,15±0,22 1,17±0,36 1,3±0,11 1-2 Leukocytes х109/l 58,3±6,2 58,6±0,45 59,3±0,27 59,7±0,64 60,6±0,28 50-60 Lymphocytes % 90,2±1,43 89,3±1,51 89,78±1,54 90,1±0,14 90,32±0,03 90-95 Neutrophils % 4,8 ± 0,3 4,7±0,64 4,8±0,42 4,8±1,6 4,9±1,11 4-6 Monocytes % 5,0±0,3 5,3±0,69 5,5±0,72 5,5±0,41 5,6±1,3 5-8 The total protein content in blood % 6,2±0,46 6,7±0,23 6,8±0,56 7,01±1,31 7,12±0,34 6-10 serum

Table 9 - Effect of non-traditional feed additive Tseofish on hematological indicators of starlet meat (n = 20)

The experimental groups 1 2 3 4 Unit of Control group Physiological norm Indicators experimental experimental experimental experimental measurement (BD) for sterlet group (1% group (2% group (3% group (4% Tseofish) Tseofish) Tseofish) Tseofish) Hemoglobin g / l 49,9±2,67 50,6±2,71 51,2±1,24 52±1,89 54,3±2,45 45-60 Erythrocytes х1012/l 0,587±0,17 0,591±0,23 0,598±0,52 0,613±0,64 0,621±0,29 0,500-0,800 Leukocytes х109l 45,7±2,8 46,12±2,31 46,7±2,67 46,9±2,79 47,5±1,42 45-50 Lymphocytes % 87,7±2,3 87,14±2,7 88,1±2,12 89,1±2,14 88,78±2,62 87-90 Neutrophils % 7,7±1,9 7,7±1,76 7,8±1,1 7,8±1,56 7,9±1,52 7,7-10

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Monocytes % 1,8±0,4 1,8±0,23 1,82±0,12 1,9±0,18 1,9±0,35 1,8-2,0 The total protein content in blood % 1,8±0,1 1,8±0,41 1,8±1,2 1,9±1,47 1,9±0,32 1,8-2,0 serum

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3.4Veterinary and sanitary assessment of the quality of fish, when used in the composition of the feed additive feed Tseofish

3.4.1 Organoleptic characteristics and indicators of freshness of fish meat in the diet added feed additive Tseofish

Veterinary-sanitary examination of fish and fish products is an integral part of the overall veterinary surveillance of fishery ponds, designed to ensure growing benign production in fish farms. The quality of the fish meat is influenced by various factors, including the environment, the conditions of fish, food, a variety of pathogens, parasites, xenobiotics, which could affect the quality and presentation of meat. Study of the effect of feed and feed additives on the organoleptic and biochemical indices of fish meat is used to evaluate the quality of the fish meat, as well as to evaluate the harmlessness and usefulness of eating fish, which was added to the feed feed additive Tseofish. Object of study for research in veterinary and sanitary examination of meat of fish (organoleptic, physical and chemical) were fish, which feed added NFA Tseofish. For sensory studies and research on the freshness of fish meat were used 2000 fish species of rainbow trout and sterlet, which under conditions of fisheries were kept in artificial pools in the diet every day 6-8 times added 1%, 2%, 3 % and 4% of the feed additive Tseofish. In sensory evaluation we had pay attention to the appearance (surface finish, natural color, knocked scales, the presence of external damage), correct cutting, and consistency (thick, it is possible, weakened but not flabby), smell (typical fresh fish). Safety of fresh fish is estimated by content of toxic elements, pesticides, in accordance with the «Hygienic requirements for quality and safety of food raw materials and food products. Sanitary norms and rules, 1997». In the sensory examination of the samples of rainbow trout and sterlet juvenile in the feed which was added 1% Tseofish scales were solid, shiny, with a pearl shade and held firmly. Skin was smooth, clean, smooth and free of bruising and mechanical damage, and was covered with slightly tarnished mucus. Eyes shining, bulging. Gills pale pink color, no smell of decay and rot. Then we did the autopsy of abdomen and internal organs of examined fish. Internal organs were located in their places, there was no noticeable bruising, authorities have not been increased in volume, and the smell was specific. Musculature was firm, supple, elastic, with pressure on the skin of a finger fossa does not remain. Fish, food which was added 1% Tseofish had specific fresh smell. When the sample boiling broth was clear, fragrant. In the sensory examination of the samples of rainbow trout and sterlet in the feed which was added 2% Tseofish noted seamless, shiny scales, skin that was smooth, clean, free of mold and mustiness, his eyes were shining, slightly sunken in the orbit. Gills were intensely red, covered with slime. Muscle tissue is dense, elastic, pressure-sensitive pit leveled for 5 seconds.

63 When the sample was not cook broth cloudy, with a specific flavor. When the sensory evaluation of rainbow trout and sterlet meat in the feed which was added 3% Tseofish noted seamless, shiny scales, no mold, which was held very firmly. The fish was covered with a thin layer of slightly tarnished mucus. The skin is smooth, shiny, shiny eyes, bulging. Gills were pale pink, slimy, with no signs of decay, unpleasant musty smell. Musculature was firm, supple, with no signs of decay, had a peculiar smell of fish. When the sample was cooking broth had transparent color, with a specific flavor. When the sensory evaluation of rainbow trout and sterlet meat, which was added to the feed of 4% Tseofish was observed seamless, shiny scales, fish was covered slightly tarnished mucus, eyes were shining. Fish had a particular smell fresh, not musty. Muscles had a dense, elastic consistency, when pressing a finger on the skin does not pit remained. When the sample by boiling the broth was cloudy and not to a specific smell of fish, without the stuffiness and bitterness. Thus, for the Sensory Evaluation of all samples of rainbow trout and sterlet, which was added to the feed daily NFA Tseofish positive results were obtained, ie organoleptic attributes all fish samples meet sanitary requirements. From physico- chemical parameters were studied: the definition of pH (potentiometric method using the device - universal ionomer I-500), reacted with copper sulphate (CuSO4), on hydrogen sulfide, peroxidase, amino-ammonium by Nessler. According to the results of organoleptic and physico-chemical studies gave sanitary assessment of fish, which was added to the feed NFA Tseofish.

64 Table 10 - Change of physico-chemical parameters of rainbow trout meat using a feed additive in feed Physico-chemical characteristics of meat reaction reaction with amino- to the reaction to reaction with Groups a 5% solution bacteriosco ammonia sample by peroxida hydrogen Nessler's рН of copper py nitrogen cooking se sulfide reagent sulfate (mg)

5-7 cocci and coli on Transparent, The broth the surface, Transparent, loss jelly-like was clear, Control * in the deep No reaction, turbidity and precipitate 1,25±0,2 with a + 6,8±0,2 layers no unchanged yellowing was was observed specific cocci and not observed smell of fish coli

4-5 cocci and coli on the surface, In broth no No reaction, Transparent, 1 Experimental Transparent, in the deep change, with staining in turbidity and (1% without 1,24±0,3 + 6,7±0,3 layers no a specific brown did yellowing was Tseofish)** flakes cocci and smell not happen not observed coli

Transparent, 3-4 cocci Transparent, 2 Experimental jelly-like and coli on The broth turbidity and No reaction, (2% precipitate the surface, 1,24±0,2 was clear, + yellowing was 6,6±0,3 unchanged Tseofish)*** was not in the deep no change not observed observed layers no

65 cocci and coli 2-3 cocci Transparent, and coli on Transparent, Broth clear, 3 Experimental jelly-like the surface, No reaction, turbidity and liquid, (3% precipitate in the deep 1,25±0,3 + no staining yellowing was 6,7±0,2 without Tseofish)**** was not layers no occurred not observed flakes observed cocci and coli 2-3 cocci The broth Reaction Transparent, and coli on Transparent, was clean, absent, 4 Experimental jelly-like the surface, turbidity and transparent staining in (4% precipitate in the deep 1,23±0,2 + yellowing was 6,6±0,1 with a brown or Tseofish)***** was not layers no not observed specific dark brown observed cocci and smell not occurred coli *- clinically healthy fish ** - fish that received with feed 1%Tseofish ****- fish that received with feed 3%Tseofish *** - fish that received with feed 2%Tseofish ***** - fish that received with feed 4%Tseofish

66 The results of studies of physical and chemical parameters of the samples of rainbow trout and sterlet, which was added to the feed NFA Tseofish are shown in Tables 10 and 11. Table 10 shows that the pH of the fish meat of the test groups of rainbow trout was in the pH of fresh meats, and does not differ from the pH of the fish meat of the control groups. If the pH of the control group of fish meat was equal to an average of 6,8 ± 0,2, then 1 test group of fish (fish that received with 1% Tseofish food) we observe a decrease in pH to 6,7 ± 0,3. In the 2 experimental group (fish, half-shell with the feed of 2% Tseofish) his record is still on the decline and is 6,6 ± 0,3, whereas in the experimental group 3 (fish that received with food 3% Tseofish) its value again varies within the range 6,7 ± 0,2. And in the 4 experimental group (fish that received with feed 4% Tseofish) rainbow trout, the pH is lowered again to 6,6 ± 0,1. Muscle tissue of fresh fish is slightly acidic (pH 6.5-6.8). By moving the pH of fish meat to the alkaline side watching the accumulation of ammonia in it and other amines, which in turn is an indicator of the intensity of development of microorganisms that cause spoilage. With the development in the body of various pathological processes observed shift in pH or the acid or alkaline side. When a deviation from the normal pH of the enzyme activity is significantly reduced, this ultimately leads to death of the organism. Thus, in our studies, pH values of rainbow trout meat in the experimental groups were normal and ranged from 6,6 ± 0,1 to 6,8 ± 0,2. Studies have found that by setting the reaction with 5% aqueous solution of copper sulphate filtrate meat and fish in the control and experimental groups is transparent, clean, without flakes, without the formation of a gelatinous precipitate. When microscopy smears revealed that the microflora in the experimental groups and control groups of rainbow trout in the deep layers is absent, on the surface of the control group - 5-7 cocci and rods, in the experimental group 1 - 4-5, in the test group 2 - 3 -4, in 3 and 4 of the experimental group - 2-3 cocci and rods. Significant reduction in bacterial numbers is likely due to the fact that zeolites possess antibacterial properties, i.e. inhibit the growth of bacteria. To enhance the bactericidal properties of zeolite-containing supplements been modified elements that suppress the growth of bacteria. For inoculation is using copper or silver. These ions are known and widely used to combat various bacteria. These elements are most suitable because they are most visible and have lower toxicity to animals as compared with other elements. They are used in human and veterinary medicine for the treatment of various human diseases and animals.

67 Table 11 - Change of physico-chemical parameters of sterlet meat using a Tseofish feed additive in feed Physico-chemical characteristics of meat reaction reaction with amino- to the reaction to reaction with Groups a 5% solution bacteriosco ammonia sample by peroxid hydrogen Nessler's рН of copper py nitrogen cooking ase sulfide reagent sulfate (mg)

Transparent, Broth Extract without 20 cocci on without colouration in Transparent, flakes, jelly- the surface, Control * changes, brownish yellowing like 1 bacteria 1,24±0,1 + 6,7±0,4 with a colour did not was not precipitate in the deep specific happen, no observed was not layers smell reaction observed Extract Broth was Broth is colouration in 15 on the transparent, transparent, brownish or Transparent, 1 Experimental surface, no without jelly- 1,23±0,3 with a + dark brown turbidity was 6,8±0,3 (1% Tseofish)** one in the like specific colour did not not observed deep layers precipitate smell happen, no reaction Not muddy, 12 on the The broth 2 Experimental No reaction, Transparent, jelly-like surface, no was (2% 1,23±0,2 + extract was turbidity was 6,7±0,2 precipitate is one in the transparent, Tseofish)*** not coloured not observed not observed deep layers no change 3 Experimental Not muddy, 10 cocci on The broth No reaction, Transparent, (3% jelly-like the surface, 1,22±0,3 was + the the extract turbidity and 6,7±0,3 Tseofish)**** precipitate is no one in transparent colour yellowing

68 not observed the deep with a without was not layers specific changes observed smell of fish Transparent, The broth Reaction is without the was clear, Transparent, 8 on the absent, 4 Experimental flakes, jelly- transparent, turbidity and surface, no colouration in (4% like 1,23±0,1 without + yellowing 6,7±0,2 one in the dark brown Tseofish)***** precipitate flakes, with was not layers did not was not a specific observed happen observed smell *- clinically healthy fish ** - fish that received with feed 1%Tseofish ****- fish that received with feed 3%Tseofish *** - fish that received with feed 2%Tseofish ***** - fish that received with feed 4%Tseofish

69 It was revealed that the number of amino-ammonia nitrogen in the experimental groups averaged 1,24 ± 0,25 mg, whereas in the control group it was 1,25 ± 0,2 mg. Consequently the number of amino-ammonia nitrogen was normal. When setting the sample boiling following results were obtained: the broth filtrate of fish in the control group was transparent, fragrant, with a specific smell of fish. In all experimental groups of rainbow trout fish broth filtrate was transparent, clean, and free of flakes and extraneous odours. It is found that when setting reaction to peroxidase in samples of fish, which feed, 1%, 2%, 3%, 4% Tseofish hood acquired a blue-green color, which after 1 minute has passed in brownish-brown colour, indicating the freshness of samples meat. That is, the peroxidase reaction was positive in all experimental groups. The positive reaction was observed also in the control group. When settling the reaction to hydrogen sulfide samples, all samples, as well as the control and experimental groups meet sanitary standards, since there was no reaction, droplets deposited on the meat is colored dark brown. Determination of Nessler’s number showed that the filtrate obtained from the fish as the experimental and control groups were transparent without opacities and yellowing. This confirms that the Nessler’s number not exceed 1.0, which implies the conclusion that all fish samples were fresh. From the table date it can be concluded that all fish in the diet was added 1%, 2%, 3%, 4% Tseofish feed additive according to the degree of freshness responded all sanitary requirements and fish meat suitable for human consumption.

3.4.2 The chemical composition and nutritional value of fish meat when using a feed additive Tseofish

The chemical composition of fish 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 fish is not permanent. It essentially depends on the species, physiological state, age, sex, habitat, and other factors. Determination of the chemical composition and nutritional value of fish meat is one of the important components of the veterinary-sanitary examination, because the ratio of moisture, protein, fat and minerals depends on the nutritional value of fish and its physiological role as a source of biologically active substances. Addition of different feed additives in fish diet not only improves the aesthetic appearance of the fish products, but also increases periods of storage and increases the content of vitamins, minerals, nutrients. In this regard, an important issue is to study the chemical composition of fish meat in the diet was added Tseofish feed additive for the content of mineral elements and organic compounds of different classes. The objects of our study were 2 kinds of valuable fish species, the most common in the majority of artificial reservoirs in Kazakhstan, which are the objects of amateur and commercial fishing and, accordingly, are often offered for sale: rainbow trout and sterlet.

70 Determination of moisture, protein, fat and minerals in fish meat was carried out by standard methods. Table 12 shows the results of concentration of protein, fat and ash in rainbow trout muscle of control and experimental groups (Figure 5). Fish proteins (5-25% more) constitute about 85% of the amount of nitrogenous substances and biological value not inferior meat proteins warm- blooded animals. Fish proteins are high-grade, and are presented in a basic, simple proteins, which are divided into water-soluble (myoglobin, globulin-X mioalbumin); salt-soluble (meozin, actin, aktomeozin, tropomeozin); not soluble in water and salt solutions, but soluble in alkali and acid complex proteins, nucleoproteins, fosfoproteidy, glucoproteins. Proteins belonging to the muscular tissue contains predominantly in the form of colloidal gels and sols. This determines the instability and volatility properties of proteins. When evaluating the quality of fish in our experiments it was found that the protein content in the fish which diet was added Tseofish feed additive was higher compared with control group. The amount of protein in the meat of the experimental groups increased in the first experimental group by 0,81% and in the second experimental by 1,25%, in the third by 1,93%, in the fourth experimental group by 3,16%, respectively, compared with the control group. The results of these studies indicate a high content of protein in the meat of fish fed the feed additive Tseofish that allows us to conclude about high nutritional value and prospects of the use of the feed additive in the production of high quality fish products. The moisture content in the experimental group of fish receiving the feed additive in an amount of 1%, 2%, 3%, 4% was normal. A slight increase in moisture content of the meat of the first and second experimental groups was observed, which rose by 0,23%, in the third experimental group by 0,1%, in the fourth by 0,2%. Researches carried out on the moisture content indicate that Tseofish feed additive in an amount of 1, 2, 3, and 4% had no significant impact on the amount of meat moisture.

Table 12 - Chemical composition and nutritional value of rainbow trout in the control and experimental groups when using the feed additive Tseofish

Indicators Control Experimental groups (n=20) Units of group 1% 2% 3% 4% measureme (n=20) nt Protein 16,00±0,17 16,7±0,32 17,08±0,54 17,67±0,28 18,73±0,36 g/110g Lipids 5,30±0,11 5,40±0,14 5,35±0,11 5,45±0,10 5,5±0,21 g/110g Moisture 73,40±1,15 73,97±1,21 73,99±1,14 73,85±1,64 74,05±1,34 g/110g

71 Ash g/110g 1,43±0,063 1,43±0,87 1,44±0,24 1,45±0,52 1,45±0,41 Digestibilit 98,57 98,67 98,77 98.57 97.65 y (%) Energy 112,00±2,61 115,60±3,6 113,35±4,1 114,45±3,6 115,28±4,2 value kcal / 7 4 2 5 100g Average value of 20 samples ± standard deviation.

The fat content of fish meat of the experimental group increased by 0,37%, 0,18%, 0,55, 0,74% respectively compared to the control group. The ash content in the both groups was practically at the same level (in the control group was 1,43 in the first test group was 1,43 g / 100 g). Only in the third and fourth test group was noted insignificant increase in the amount of ash by 0,27%. The presented values are favorably comparable with published reports on different salmonid species (Тести и соавт. 2006). Also, Gonzalez et al. [37] reported higher lipid content (6.55%) and lower protein content (16.04%) in rainbow trout (O. mykiss) as compared to the findings of the present study. Digestibility of feed with unconventional feed additive Tseofish in the experimental groups of rainbow trout remained in normal (within 97,65-98,67%), therefore, feed with feed additive Tseofish well digested by the body of the fish. The energy value of rainbow trout meat while using a feed additive Tseofish increased in the experimental groups when added to feed 1 and 4% of the feed additive, probably due to the fact that at these concentrations in fish is better absorbed this feed additive.

Table 13 - Chemical composition and nutritional value of sterlet in the control and experimental groups when using the feed additive Tseofish

Indicators Control Experimental groups (n=20) Units of group 1% 2% 3% 4% measuremen (n=20) t Protein 17,05±0,21 16,7±0,12 17,08±0,43 17,54±0,22 18,06±0,47 g/110g Lipids 6,50±0.,26 5,79±0,09 6,79±0,11 7,24±0,33 7,16±0,51 g/110g Moisture 77,70±1,17 75,67±1,41 77,89±1,32 77,92±1,56 78,02±1,24 g/110g Ash g/110g 1,5±0,33 1,47±0,79 1,56±0,45 1,6±0,22 1,5±0,41 Digestibility 98,84 98,91 97,99 98,90 98,65 (%) Energy value 88,00±2,78 89,60±2,90 88,35±3,19 90,45±3,54 90,97±3,22 kcal / 100g Average value of 20 samples ± standard deviation.

72 Table 13 shows the chemical composition and nutritional value of sterlet at the control and experimental groups when using non-traditional feed additive Tseofish in the diet. By results of researches, it was found that the protein content in the experimental groups significantly increased compared with control groups. For example, if samples of fish 1 experimental group (1% feed additive Tseofish was added in the diet) protein amount was equal to 19,7%, then in the third, and in the fourth experimental group is significantly increased to 20,3% and 20,9% respectively. In the control group, protein amount was equal approximately to 19,7%. Also was noted an increase in the amount of fat in the experimental groups compared with the control group. If the amount of fat was equal to 19,4% in control group, it was markedly increased to 20,3% and 21,6% in the second and third group. And fat content was winding down again in the fourth experimental group of sterlet, as in the second group to 21,3%(Figure 6). The moisture and ash content in the experimental and control groups did not change. If the moisture content of the control group reached to 20,0% in the experimental groups was equal in the first – 19,5%, in the second – 20,1%, in the third – 20,1%, in the fourth – 20,1%, respectively. Ash content also remained unchanged, all parameters were normal. Its number is equal to 19,6% in the control group, 19,2% in the first experimental group, 20,4% in the second experimental group, 20.9% in the third group, 19,2% in the fourth group.

Figure 5 - The chemical composition and nutritional value of rainbow trout, using the feed additive Tseofish (g / 100 g) in the diet

73

Figure 6 - The chemical composition and nutritional value of sterlet, using the feed additive Tseofish (g / 100 g) in the diet

Results of the study of starlet meat were showed that the using of the feed additive Tseofish in the diet of fish did not adversely affect the chemical composition of meat. The digestibility of the feed additive by sterlet also remained within normal limits, as in the experimental groups of rainbow trout. The energy value analysis results showed an increase of this indicator in fish of the first, third and fourth experimental groups. From this it follows that the more contain of Tseofish feed additive in the diet to influence the fish body better, the energy value increases in proportion to the content of the feed additive Tseofish in feed. Thus, in a study of the chemical composition and nutritional value of meat of valuable fish species (rainbow trout and sturgeon) in the feed, which was introduced non-traditional feed additive Tseofish had been found that this feed additive is completely harmless, has no negative effects on the chemical composition of fish meat in the experimental groups, while in the experimental groups were significantly improving some indicators: protein in the first experimental group by 0,81%, in the second experimental by 1,25%, in the third - by 1,93%, in the fourth experimental group by 3,16%, fat by 0,37%, 0,18% , 0,55%, and 0,74% respectively.

74 3.4.3 Amino acid composition of fish meat in the diet when using NFA Tseofish

The quality of the fish protein evaluated on the basis of the amino acids contained in it. The most important amino acids are exogenous amino acids, which are indispensible for the human body. The amino acid content (g/100 g amino acid) in the rainbow trout and sterlet from the control and experimental groups has been analyzed, and the results are shown in Table 14, 15 and diagrams 7, 8. In this study, some amino acids exhibited a significant difference of content between the groups. Differences in the types and amounts of amino acids in fish tissues have been attributed to the location, size of fish, age, food, reproductive status and season.

Table 14 - Content of amino acids in muscle tissue of rainbow trout when using as a feed additive Tseofish (control and experimental groups, g / 100 g muscle)

Amino acids Control Experimental group group (n=20) (n=20) 1% 2% 3% 4% Threonine 0,72±0,03 0,87±0,05 0,86±0,04 0,89±0,04 0,95±0,06 Valine 0,89±0,07 0,95±0,07 0,95±0,08 0,99±0,06 1,09±0,09 Isoleucine 0,80±0,12 0,68±0,09 0,71±0,07 0,80±0,10 0,78±0,08 Leucine 1,69±0,21 1,79±0,20 1,87±0,18 1,92±0,22 2,01±0,18 Tyrosine 0,54±0,12 0,47±0,10 0,57±0,12 0,54±0,15 0,61±0,13 Phenylalanine 0,89±0,05 0,91±0,07 0,99±0,08 0,94±0,04 1,01±0,07 Cysteine 0,25±0,02 0,27±0,01 0,29±0,02 0,31±0,02 0,33±0,03 Methionine 0,45±0,04 0,55±0,06 0,52±0,07 0,51±0,04 0,55±0,07 Tryptophan 0,27±0,03 0,18±0,01 0,21±0,02 0,27±0,03 0,21±0,02 Lysine 1,20±0,21 1,40±0,22 1,40±0,18 1,20±0,28 1,40±0,19 ΣEAA, 7,70 8,07 8,37 8,37 8,94 Total essential amino acids Aspartic acid 1,05±0,21 1,15±0,17 1,25±0,23 1,25±0,31 1,15±0,28 Histidine 0,56±0,17 0,58±0,14 0,65±0,12 0,66±0,17 0,65±0,11 Arginine 1,46±0,32 1,50±0,38 1,5±0,44 1,46±0,28 1,5±0,24 Serine 0,55±0,07 0,45±0,08 0,45±0,07 0,55±0,05 0,45±0,04 Glutamic acid 3,05±0,23 3,27±0,18 3,28±0,19 3,25±0,19 3,28±0,21 Proline 0,57±0,04 0,46±0,04 0,46±0,04 0,57±0,05 0,64±0,06 Glycine 0,82±0,08 0,60±0,09 0,60±0,10 0,86±0,11 0,76±0,09 Alanine 0,94±0,09 1,00±0,08 1,11±0,12 0,94±0,10 1,26±0,13 ΣNEAA, 9 9,01 9,3 9,54 9,69 Total essential amino acids

75 ΣEAA/ΣNEAA 0,86±0,06 0,89±0,07 0,90±0,06 0,88±0,07 0,92±0,08 The most exogenous amino acids were found in the rainbow trout fed with 4% zeolite feed – 8.94 g of these amino acids were found in 100 g of the muscle tissue. The daily demand for the amino acids required for an adult human weighing 70 kg is equal to 5.59 g [44]. The results of the studies indicated that 100 g of the fish muscle tissue orom each experimental group covered the ingredient's daily demand of an adult human. A glutamic acid (containing 3.25-3.28%) was present in the highest amount among the amino acids measured in all the experimental groups. Similar results for the glutamic acid was reported in the rainbow trout (O. mykiss), an Atlantic salmon (Salmo salar), a channel catfish (Ictalurus punctatus) [45], Oreochromis niloticus, Tilapia zilli, Sarotherodon galileaus, Clarias anguillaris, Clarias gariepinus and Heterobranchus longifilis [46], and a Beluga Sturgeon (Huso huso) [47]. Glutamine from muscles serves as an important carrier of ammonia (nitrogen) to the immune system [48]. The most important amino acids in terms of nutritional value that cannot be synthesized by the human body and must be supplied in diet are: lysine, methionine, and cysteine. Histidine is another valuable amino acid for the human body and belongs to the group of relatively exogenous amino acids that are produced in a human body. However under certain conditions, e.g. a rapid growth or a disease, its amount is insufficient, and must be supplied in diet. A particularly advantageous increase of the content was noted for lysine, methionine, and cysteine in the group of the fish fed with the addition of zeolite. The highest content was noted for the group of the fish fed with the addition of 4% zeolite. In this study, the content of the essential amino acids in the rainbow trout fed with RGM-2M enriched with zeolite was higher (8,07–8,94%) than the content in the control group (7,7%). The nonessential amino acid content was also higher in the rainbow trout from the experimental groups (9,01–9,69%) than in the trout from the control group (9,00%) These content levels were the evidence that the rainbow trout fed with RGM 2M/zeolite were a very good source of the amino acids. It was significant to note that the rainbow trout contained a broad variety of the amino acids and their isomers, as well as a particularly high proportion of EAA. There were higher levels of the amino acids in the experimental fish than in the control group. The most significant increases in the experimental groups in comparison to the control group were observed for the leucine and the lysine. The significant decreases were observed for the tryptophan and the glycine. The proportion of those essential amino acids to the non-essential amino acids was greater for the experimental group (0,89–0,92) than the control group (0,85). In the study, this proportion was greater than it had been stated in other works: it was 0,78 for Huso huso [47], 0,77 for a sea bream (Pagrus major), 0,77 for a mackerel (Scomber japonicus), 0,71 for a mullet (Mugil cephalus), 0,69 for a sardine (Sardina melonosticta), 0,74 for a herring (Clupea pallasi), and 0,75 for a chum salmon (Oncorhynchus keta). Dezhabad et al. [49] had reported that the

76 proportion of the essential amino acids to the non-essential amino acids for the three species: Rutilus frisii, Hypophthalmicthys molitri and Oncorhynchus mykiss, ranged from 1,03 to 1,19. Approximately the same amount of essential amino acids has a fourth experienced group of sterlet which feed was added feed additive Tseofish. In the study of fish amino acid content of experimental groups can be seen raising the total number of essential and non-essential amino acids in the experimental groups compared with the control group. Especially essential and nonessential amino acids indicators reach their maximum values at the fourth experimental group of fish. If rainbow trout of 4 experimental group ΣEAA reached 8,94 g, then it was 8,05 in starlet. The maximum content of the total number of essential amino acids is also observed in the fourth experimental group of fish. If ΣNEAA at rainbow trout of fourth experimental group reached 9,69 g, then at the sterlet it was 8,56. The difference in the content of the total number of essential and nonessential amino acids in rainbow trout and sterlet explained by the fact that they belong to a different kind, rainbow trout belongs to the kind of salmon and sterlet in their turn to the sturgeon kind.

Table 15 - Content of amino acids in muscle tissue of sterlet when using as a feed additive Tseofish (control and experimental groups, g / 100 g muscle)

Amino acids Control Experimental group group (n=20) (n=20) 1% 2% 3% 4% Threonine 0,81±0,12 0,90±0,03 0,88±0,05 0,93±0,17 0,93±0,06 Valine 0,78±0,11 0,84±0,04 0,82±0,16 0,87±0,12 0,91±0,08 Isoleucine 0,85±0,21 0,77±0,08 0,82±0,13 0,86±0,06 0,88±0,04 Leucine 1,25±0,17 1,24±0,09 1,28±0,10 1,25±0,21 1,28±0,06 Tyrosine 0,62±0,16 0,57±0,01 0,59±0,11 0,62±0,04 0,63±0,12 Phenylalanine 0,94±0,03 0,90±0,05 0,96±0,18 0,95±0,06 1,00±0,13 Cysteine 0,20±0,12 0,21±0,15 0,25±0,02 0,28±0,01 0,31±0,17 Methionine 0,42±0,02 0,40±0,06 0,44±0,14 0,51±0,04 0,49±0,08 Tryptophan 0,20±0,06 0,16±0,16 0,19±0,11 0,24±0,02 0,22±0,18 Lysine 1,26±0,27 1,40±0,18 1,39±0,14 1,32±0,23 1,40±0,07 ΣEAA, 7,33 7,39 7,62 7,83 8,05 Total essential amino acids Aspartic acid 0,85±0,18 0,90±0,22 0,88±0,15 0,97±0,19 0,95±0,21 Histidine 0,52±0,11 0,50±0,18 0,51±0,21 0,56±0,13 0,60±0,01 Arginine 1,44±0,23 1,46±0,27 1,48±0,33 1,46±0,29 1,5±0,20 Serine 0,49±0,08 0,49±0,12 0,47±0,15 0,53±0,25 0,51±0,02 Glutamic acid 2,55±0,19 2,49±0,18 2,52±0,19 2,58±0,23 2,60±0,21 Proline 0,52±0,02 0,47±0,06 0,49±0,12 0,57±0,08 0,59±0,15

77 Glycine 0,74±0,04 0,70±0,12 0,78±0,09 0,80±0,18 0,82±0,11 Alanine 0,88±0,16 0.95±0,13 0,98±0,04 0,96±0,27 0,99±0,18 ΣNEAA, 7,99 7,96 8,11 8,43 8,56 Total essential amino acids ΣEAA/ΣNEAA 0,91±0,06 0,92±0,07 0,93±0,06 0,92±0,07 0,94±0,08

Foregoing is evidence that feeding rainbow trout and sturgeon pelleted feeds with content of the feed additive Tseofish can be a good source for meet the shortfall of the amino acid composition of the fish meat.

78 2,5

2,01 2 1,92 1,87 1,79 1,69

1,5 1,41,4 1,4 Контрольная группа

1,2 1,2 1 опытная группа 2 опытная группа 1,09 0,99 0,991,01 3 опытная группа 1 0,890,95 0,950,95 0,91 0,94 0,870,86 0,89 0,89 4 опытная группа 0,8 0,8 0,72 0,78 0,680,71 0,61 0,55 0,54 0,570,54 0,55 0,51 0,52 0,5 0,47 0,45 0,31 0,29 0,33 0,27 0,27 0,250,27 0,21 0,18 0,21

0 Треонин Валин Изолейцин Лейцин Тирозин Фенилаланин Цистеин Метионин Триптофан Лизин

Figure 7 - Exogenous amino acid content in the muscle tissues of rainbow trout when used Tseofish feed additive (g/100 g)

79 3,5 3,28 3,27 3,25 3,28 3,05 3

2,5

2 Контрольная группа 1 опытная группа

1,5 1,5 1,5 2 опытная группа 1,5 1,46 1,46 3 опытная группа 1,251,25 1,26 4 опытная группа 1,15 1,15 1,11 1,05 1 1 0,94 0,94 0,82 0,86 0,76 0,65 0,64 0,66 0,65 0,57 0,6 0,58 0,55 0,55 0,57 0,6 0,56 0,45 0,5 0,45 0,45 0,46 0,46

0 Аспаргиновая кислота Гистидин Аргинин Серин Глутаминовая кислота Пролин Глицин Аланин

Figure 8 - Endogenous amino acid content in the muscle tissue when used rainbow trout feed additive Tseofish (g/100g)

80 3.4.4 Analysis of the fatty acid composition of fish meat when added to feed NFA Tseofish

Fish fats are a mixture of glycerol estera and fatty acids. An important feature is the predominance of fat in their composition of unsaturated fatty acids (84%) and the presence among them highly unsaturated with 4 - 6 double bonds, which are in the fats of land animals are not available. Unlike fat warm-blooded animals, fish fat has a liquid consistency with the specific taste and smell, and is easily digested by the human body, characterized by a high nutritional value, is a valuable source not synthesized in the body acids (linolenic, linoleic and arachidonic), which normalize lipid metabolism and promote the excretion of cholesterol. The fat in the fish body is unevenly distributed, and it depends on the kind of fish and their physiological properties. Different fish fat is concentrated in different areas of the body. Mostly it is in the subcutaneous layer and around the fins (herring, sardines, salmon), between the muscle fibers (sturgeon, herring, catfish), along the spine (flounder, halibut) in the liver and along the lateral line. The largest amount phosphatides (most studied lecithin) steridy and sterols (cholesterol) and other colorants present in the fish fat. The energy and nutritional value is significiantly depend on the fat content of fish, therefore fatness of fish is an important indicator in determining the grade of fish products. The fat content in the fish meat greatly varies between 0,5 - 30%, more preferably from 2 - 12%. It is known that the fish meat does not contain significant amounts of the lipids. However, the study of its fatty acid content is of significant interest, not from the perspective of determining its biological value, but as the indicator which points at cell abnormalities in biochemical processes. It was found that the zeolite increased mainly the level of polyunsaturated fatty acids in the fish meat. The biochemical composition may be affected by the species of the fish, environmental factors, size, age, and diet [41-43,50-52]. The fish can be a source of essential fatty acids [53]. In this study, FA contents (% of total FAs) in the rainbow trout and starlet fed with RGM-2M with zeolite from the experimental and control groups were given in Table 13, 14 and in diagrams 10, 11. After analysing of the fatty acids it was found that there were higher and lower levels of the fatty acids in the experimental fish than in the control group. The FA contents of the fish in the control and experimental groups ranged from 26,25% to 27,37% of the saturated fatty acids (SFAs), 30,04–31,29% of monounsaturated acids (MUFAs) and 35,44–36,71% of PUFAs. The lipids in the fatty muscle tissue of the trout fed with 2 and 3% zeolite feed contained the most saturated fatty acids. The majority of monoene acids were contained in the lipids of the muscle tissue of the fish fed with feed with the addition of 2 and 3% zeolite. The level of SFAs was comparable, and MUFAs was significantly lower than the level observed by Łuczyńska [54]. Most n-6 polyene fatty acids were noted in the muscle tissue of the fish fed with the 1 % zeolite feed. However this content was higher in all the experimental groups in comparison to

81 the control group. Among them, those present in the highest content in the experimental group of the fish were C18:1n9, an oleic acid (OLA, 20,08-21,72 %), C16:0, a palmitic acid (PAA, 15,34-16,22%), DHA (15,05-16,09%), C18:2 a linoleic acid (LIA 11,06-11,45%), a palmitoleic acid (PLA 5,30-5,69%), C16:1 EPA (3,86-4,21%), a stearic acid (STA 3,15-3,44%), and C14:0, and a myristic acid (MYA, 3,27-3,85%). Epidemiological studies showed that an n-3 fatty acid intake is inversely related to cancer, cardiovascular diseases [55], psychiatric disorders [56], asthma [57], bone mineral density [58] and type 2 diabetes [59]. Because of this fact, the polyunsaturated fatty acids (PUFAs) should be separated into the n-3 and n-6 fatty acids. Although the n-3 and n-6 PUFA levels in the two experimental groups (1% and 3% zeolite) were higher than in the control group, the difference was statistically significant. The muscle tissue of the trout fed with 4% zeolite feed was characterized as the richest source of EPA. The linoleic acid was dominant in the group of n-6 fatty acids, and DHA and EPA were dominant in the n-3 group. Other researchers have made similar observations [54, 60, 61]. The proportions of FAs-n3 (21,35%; 21,56–22,71% control and experimental groups) were generally higher than those of FAs-n

Table 16 - The fatty acids compositions of rainbow trout (control and experimental groups, % of total fatty acid). The content of fatty Control Experimental group (n =20) acids, group (n 100g-1 =20) 1% 2% 3% 4%

The content of saturated fatty acids 12: 0 (Lauric acid) 0,04±0,03 0,04±0,02 0,05±0,02 0,05±0,02 0,04±0,02 13: 0 (Tridecanoic acid) 0,02±0,02 0,01±0,04 0,03±0,02 0,02±0,02 0,01±0,023 14: 0 (Myristic acid) 3,27±0,06 3,85±0,07 3,76±0,03 3,72±0,03 3,45±0,02 15: 0 (Pentadecanoic 0,37±0,03 0,37±0,05 0,37±0,04 0,36±0,05 0,36±0,03 acid) 16: 0 (Palmitic acid) 15,99±0,05 16,16±0,08 16,05±0,07 16,22±0,10 15,34±0,08 17: 0 (Heptadecanoic 0,68±0,02 0,58±0,03 0,53±0,05 0,52±0,02 0,48±0,03 acid) 18: 0 (Stearic acid) 3,42±0,13 3,25±0,02 3,44±0,04 3,15±0,04 3,25±0,05 20: 0 (Arachidonic 0,22±0,03 0,21±0,02 0,16±0,02 0,23±0,02 0,21±0,02 acid) 22: 0 (Behenic acid) 1,54±0,14 1,26±0,05 1,32±0,05 1,48±0,05 1,53±0,04 C 23:0 (Tricosanoic 0,04±0,02 0,04±0,06 0,03±0,03 0,04±0,02 0,04±0,02 acid) C 24:0 (Lignoceric 1,35±0,02 1,40±0,03 1,44±0,04 1,25±0,05 1,54±0,03 acid) Total SFAs 26,94 27,17 27,18 27,04 26,25 Monounsaturated fatty acids

82 C 14:1 (Myristoleic 0,23±0,06 0,23±0,04 0,24±0,03 0,23±0,03 0,21±0,02 acid) C 16:1 (Palmitoleic 5,30±0,02 5,29±0,08 5,25±0,05 5,36±0,04 5,95±0,02 acid) C 17:1 (cis 10 – 0,26±0,06 0,23±0,02 0,21±0,02 0,23±0,02 0,35±0,03 heptadecenoic acid) C 18:1 n9 (Oleic acid) 20,98±0,09 20,27±0,11 21,72±0,10 21,52±0,07 20,08±0,08 C 20:1 (cis -11- 2,43±0,02 2,41±0,05 2,35±0,04 2,41±0,03 2,41±0,02 eicosenoic acid) C 24:1 (Nervonic acid) 1,46±0,02 1,61±0,06 1,52±0,06 1,53±0,02 1,48±0,02 Total MUFAs 30,66 30,04 31,29 31,28 30,48 Polyunsaturated fatty acids C 18:2 n6 (Linoleic 11,06±0,02 11,11±0,02 11,24±0,03 11,35±0,02 11,45±0,03 acid) C 18:3 n6 (γ-linolenic 0,29±0,03 0,42±0,02 0,35±0,02 0,28±0,02 0,26±0,02 acid) C 18:3 n3 (Linolenic 1,86±0,02 1,99±0,05 1,89±0,04 1,94±0,03 1,86±0,04 acid) C 20:2 (cis-11,14- 0,58±0,02 0,50±0,03 0,45±0,04 0,55±0,04 0,65±0,04 eicosadienoic acid) C 20:3 n6 (cis-8,11,14- 0,24±0,07 0,23±0,07 0,24±0,07 0,21±0,06 0,23±0,07 eicosatrienoic acid) C 20:3 n3 (cis- 0,58±0,02 0,58±0,02 0,51±0,02 0,56±0,02 0,58±0,02 11,14,17-eicosatrienoic acid) C 20:4 n6 (Arachidonic 0,87±0,02 0,69±0,07 0,79±0,07 0,71±0,06 0,87±0,07 acid) C 20:5 n3 (cis- 3,86±0,02 4,05±0,03 4,11±0,03 4,12±0,03 4,21±0,03 5,8,11,14,17- eicosapentaenoic acid) EPA C 22:2 (cis 13,16 – 1,05±0,06 1,05±0,02 1,02±0,02 1,02±0,02 1,06±0,02 docosadienoic acid) C 22:6 n3 (cis- 15,05±0,02 16,09±0,02 15,05±0,02 15,35±0,02 15,34±0,02 4,7,10,13,16,19- docosahexaenoic acid) DHA Total PUFAs 35,44 36,71 35,65 36,09 36,51 PUFAs/SFAs 1,32±0,02 1,35±0,02 1,31±0,05 1,33±0,05 1,39±0,03

The biochemical composition may be affected by the species of the fish, environmental factors, size, age, and diet [41-43, 50-52]. The fish can be a source of essential fatty acids [53]. In this study, FA contents (% of total FAs) in the rainbow trout fed with RGM-2M with zeolite from the experimental and control groups were given in Table 16. After analysing of the fatty acids it was found that there were higher and lower levels of the fatty acids in the experimental fish than in the control group.

83 The FA contents of the fish in the control and experimental groups ranged from 26,25% to 27,37% of the saturated fatty acids (SFAs), 30,04–31,29% of monounsaturated acids (MUFAs) and 35,44–36,71% of PUFAs. The lipids in the fatty muscle tissue of the trout fed with 2 and 3% zeolite feed contained the most saturated fatty acids. The majority of monoene acids were contained in the lipids of the muscle tissue of the fish fed with feed with the addition of 2 and 3% zeolite. The level of SFAs was comparable, and MUFAs was significantly lower than the level observed by Łuczyńska [54]. Most n-6 polyene fatty acids were noted in the muscle tissue of the fish fed with the 1 % zeolite feed. However this content was higher in all the experimental groups in comparison to the control group. Among them, those present in the highest content in the experimental group of the fish were C18:1n9, an oleic acid (OLA, 20,08–21,72 %), C16:0, a palmitic acid (PAA, 15,34–16,22%), DHA (15,05–16,09%), C18:2 a linoleic acid (LIA 11,06–11,45%), a palmitoleic acid (PLA 5.30–5.69%), C16:1 EPA (3,86–4,21%), a stearic acid (STA 3,15–3,44%), and C14:0, and a myristic acid (MYA, 3,27–3,85%). Epidemiological studies showed that an n-3 fatty acid intake is inversely related to cancer, cardiovascular diseases [55], psychiatric disorders [56], asthma [57], bone mineral density [58] and type 2 diabetes [59]. Because of this fact, the polyunsaturated fatty acids (PUFAs) should be separated into the n-3 and n-6 fatty acids. Although the n-3 and n-6 PUFA levels in the two experimental groups (1% and 3% zeolite) were higher than in the control group, the difference was statistically significant. The muscle tissue of the trout fed with 4% zeolite feed was characterized as the richest source of EPA. The linoleic acid was dominant in the group of n-6 fatty acids, and DHA and EPA were dominant in the n-3 group. Other researchers have made similar observations [54, 60, 61]. The proportions of FAs-n3 (21,35%; 21,56–22,71% control and experimental groups) were generally higher than those of FAs-n6 (12,46%; 12,45 12,81%). The UK Department of Health recommends an ideal n6/n3 ratio of 4.0 at maximum [62]. Values higher than the maximum value are harmful to health and may promote cardiovascular diseases [63]. In this study, the n6/n3 ratio was found to be 0.55–0.59 in all the experimental groups. The recommended minimum value of the PUFAs/SFAs ratio is 0,45 [62], which is lower than the values of 1,32 and 1,31–1,39 from the control group and the experimental groups treated with RGM-2M and additives. DHA/EPA ratio ranged from 0,72 to 6,89 in some fresh water fish species [64] and it was equal to 1,56 in the rainbow trout [65]. In this study, the ratio of DHA/EPA in the rainbow trout fed with RGM-2M enriched in 1% zeolite was found to be 3,97 and was greater than in the control group (3,90). In other groups, this ratio was lower and amounted to 3,64–3,73. On the basis of the conducted analysis no single correlation could be found between the content of individual fatty acids and the percentage of zeolite's addition to the feed. Undoubtedly, the addition of the zeolite had an influence on

84 the profile of fatty acids in lipids in the muscle tissue of rainbow trout, and it also increased the content of n-3 and n-6 polyene fatty acids advantageously.

Table 17 - The fatty acids compositions of sterlet (control and experimental groups, % of total fatty acid)

The content of fatty Control Experimental group (n =20) acids, group (n 100g-1 =20) 1% 2% 3% 4%

The content of saturated fatty acids 12: 0 (Lauric acid) 0,03±0,02 0,03±0,03 0,03±0,02 0,04±0,06 0,04±0,01 13: 0 (Tridecanoic acid) 0,02±0,02 0,02±0,06 0,03±0,01 0,03±0,02 0,02±0,021 14: 0 (Myristic acid) 3,20±0,09 3,43±0,03 3,55±0,21 3,47±0,02 3,33±0,03 15: 0 (Pentadecanoic 0,30±0,03 0,30±0,05 0,31±0,09 0,30±0,02 0,31±0,06 acid) 16: 0 (Palmitic acid) 15,09±0,07 16,10±0,10 16,12±0,07 16,28±0,12 15,87±0,20 17: 0 (Heptadecanoic 0,55±0,03 0,49±0,02 0,46±0,07 0,42±0,09 0,44±0,03 acid) 18: 0 (Stearic acid) 3,75±0,09 3,67±0,03 3,79±0,10 3,71±0,14 3,77±0,06 20: 0 (Arachidonic 0,26±0,02 0,21±0,04 0,23±0,03 0,21±0,02 0,25±0,01 acid) 22: 0 (Behenic acid) 1,47±0,08 1,33±0,03 1,39±0,03 1,44±0,03 1,48±0,02 C 23:0 (Tricosanoic 0,03±0,01 0,03±0,02 0,02±0,03 0,03±0,02 0,03±0,02 acid) C 24:0 (Lignoceric 1,28±0,03 1,32±0,03 1,33±0,02 1,29±0,03 1,38±0,04 acid) Total SFAs 25,98 26,93 27,26 27,22 26,92 Monounsaturated fatty acids C 14:1 (Myristoleic 0,20±0,02 0,21±0,03 0,21±0,02 0,22±0,03 0,21±0,02 acid) C 16:1 (Palmitoleic 5,80±0,01 5,76±0,06 5,78±0,03 5,81±0,04 5,83±0,05 acid) C 17:1 (cis 10 – 0,20±0,03 0,21±0,02 0,20±0,04 0,22±0,02 0,24±0,03 heptadecenoic acid) C 18:1 n9 (Oleic acid) 20,31±0,03 20,12±0,06 21,02±0,07 21,22±0,07 21,05±0,10 C 20:1 (cis -11- 2,25±0,03 2,31±0,06 2,27±0,03 2,36±0,02 2,29±0,03 eicosenoic acid) C 24:1 (Nervonic acid) 1,37±0,03 1,42±0,05 1,39±0,02 1,51±0,03 1,45±0,03 Total MUFAs 30,13 30,03 30,87 31,34 31,07 Polyunsaturated fatty acids C 18:2 n6 (Linoleic 10,21±0,04 10,01±0,02 11,13±0,02 11,27±0,04 11,38±0,02 acid) C 18:3 n6 (γ-linolenic 0,22±0,02 0,30±0,03 0,31±0,02 0,26±0,03 0,30±0,02 acid) C 18:3 n3 (Linolenic 1,78±0,03 1,84±0,04 1,80±0,02 1,78±0,01 1,89±0,02

85 acid) C 20:2 (cis-11,14- 0,48±0,03 0,39±0,02 0,43±0,03 0,51±0,03 0,53±0,04 eicosadienoic acid) 0,21±0,09 0,20±0,03 0,24±0,12 0,22±0,05 0,21±0,10 C 20:3 n6 (cis-8,11,14- eicosatrienoic acid) C 20:3 n3 (cis- 0,51±0,03 0,53±0,04 0,56±0,03 0,58±0,01 0,57±0,04 11,14,17-eicosatrienoic acid) C 20:4 n6 (Arachidonic 0,78±0,03 0,67±0,08 0,79±0,10 0,76±0,03 0,79±0,04 acid) C 20:5 n3 (cis- 3,60±0,05 3,78±0,02 3,69±0,03 3,75±0,02 3,81±0,05 5,8,11,14,17- eicosapentaenoic acid) EPA C 22:2 (cis 13,16 – 1,16±0,08 1,17±0,04 1,03±0,03 1,06±0,03 1,16±0,05 docosadienoic acid) C 22:6 n3 (cis- 15,11±0,03 16,10±0,03 15,77±0,02 15,31±0,04 15,43±0,04 4,7,10,13,16,19- docosahexaenoic acid) DHA Total PUFAs 34,06 34,99 35,75 34,68 36,07 PUFAs/SFAs 1,13±0,02 1,16±0,02 1,14±0,05 1,13±0,05 1,16±0,03 While investigating fatty acid content of sterlets’ meat the feed which was added nontraditional feed additive Tseofish observed changes in the content of saturated fatty acids, especially: 12:0 lauric acid, 14:0 myristic acid, 16:0 palmitic acid, and 24:0 lignoceric acid. Contents of the aforementioned fatty acids increased significantly in the experimental groups compared with the control group, and their number reaches a maximum value at the fourth experimental group. We observed an increase of saturated fatty acids in the total amount in sterlet meat of experimental groups compared with the control group. In the first experimental group the total amount of saturated fatty acids was 26,93%, in the second experimental group the amount thereof is in the range of 27,26%, in the third experimental group - 27,22% in the fourth experimental group - 26,92%. Saturated fatty acid content of sterlet was less than 25,98% at the control group. Thus, in our study, the percentage of fatty acids in the flesh of rainbow trout was higher than in the conclusions of Saglyk Aslan et al. The results showed that the fatty acid is dependent on density of the feed and feed additives. And also when comparing our results with the results of the research study obtained Danabas D., notable differences in the content of fatty acids in the meat of the fish were noted. Consequently, alternative feed additive Tseofish when used as part of the fish feed has no significant negative impact on the fatty acid composition of fish meat. In addition, was observed increase in the content of polyunsaturated fatty acids, but at the same time, their level is not too high. A disadvantage of these fatty acids in the body leads to a deficiency of energy in the body, depletion, the development of various diseases of the gastrointestinal tract.

86 90

15,34 80

70 16,22 60

50 16,05

40 4 опытная группа 3 опытная группа 30 16,16 2 опытная группа 3,45 20 3,25 1 опытная группа 3,72 0,36 15,99 3,15 Контрольная группа 0,01 3,76 0,48 10 0,36 3,44 1,54 0,02 0,52 1,53 3,85 0,37 3,25 0,23 0,21 1,48 0,04 1,25 0,05 0,03 0,53 1,32 1,44 0,16 1,26 0,04 1,4 0 0,050,04 0,01 3,27 0,37 0,58 3,42 0,21 1,54 0,03 0,68 0,22 0,04 1,35 0,040,04 0,02 0,37 0,04

Figure 9 - The content of saturated fatty acids in the meat of rainbow trout feed used in the composition of the feed additive Tseofish (g / 100g)

87 120

20,08 100

21,52 80

21,72 60

20,27 4 опытная группа 40 3 опытная группа 5,95 2 опытная группа 5,36 20,98 20 2,41 1 опытная группа 5,25 5,29 2,41 1,48 Контрольная группа 0,21 0,35 0,23 1,53 0,23 2,35 0,23 0,23 5,3 0,26 2,41 1,52 0 0,23 0,21 1,61 0,24 2,43 1,46

Figure 10 - Monosaturated fatty acid in the meat of rainbow trout when used Tseofish feed additive (g / 100 g)

88 90

80 15,34

70 15,35 60 11,45 50 11,35 15,05 40 11,24 16,09 30 11,11 4,21 20 4 опытная группа 1,86 4,12 15,05 10 11,06 0,26 0,87 4,11 1,06 1,05 3 опытная группа 1,94 0,65 0,23 0,58 4,05 1,02 1,89 0,56 0,71 1,02 0,280,42 1,99 0,55 0,21 0,69 3,86 2 опытная группа 0 0,35 0,450,5 0,24 0,23 0,51 0,58 0,79 1,05 0,29 1,86 0,58 0,87 0,58 0,24 1 опытная группа Контрольная группа

Figure 11 - The content of polyunsaturated fatty acids in the meat of rainbow trout when used a feed additive Tseofish (g /100 g)

89 90

15,87 80

70 16,28 60

50 16,12

40 4 опытная группа

30 16,1 3 опытная группа 2 опытная группа 3,77 20 3,33 1 опытная группа 3,47 15,09 3,71 Контрольная группа 10 3,55 0,3 3,79 1,48 0,3 0,42 3,67 1,38 3,43 1,44 1,29 0,04 0,020,03 0,44 3,75 0,25 0,21 1,39 1,33 0,04 0,02 3,2 0,31 0,3 0,460,49 0,23 1,33 0,030,03 1,32 0 0,030,030,03 0,02 0,31 0,55 0,21 0,030,02 1,28 0,03 0,26 1,47 0,03

Figure 12 - The content of saturated fatty acids in meat starlet when used a feed additive Tseofish (g/100 g)

90 120

21,05 100

21,22 80

60 21,02

4 опытная группа 40 20,12 3 опытная группа 5,83 2 опытная группа 5,81 20,31 20 1 опытная группа 5,78 2,29 0,22 1,45 5,76 0,24 2,36 Контрольная группа 0,21 0,20,21 2,27 0,22 0,21 5,8 2,31 1,511,39 0,21 0,2 0,2 2,25 1,42 0 1,37

Figure 13 - Monounsaturated fatty acid content in meat sterlets when used Tseofish feed additive (g/100 g)

91 90

80 15,43 70 15,31 60 11,38 50 15,77 11,27 40 11,13 30 16,1 4 опытная группа 3 опытная группа 20 10,01 3,81 3,75 15,11 2 опытная группа 1,89 10 10,21 3,69 1,16 0,3 1,78 0,79 3,78 1,06 1 опытная группа 0,26 1,81,84 0,53 0,58 0,76 0,79 3,6 1,17 0,3 0,31 1,78 0,51 0,39 0,2 0,22 0,530,570,56 1,03 0 0,22 0,43 0,48 0,24 0,21 0,51 0,670,78 1,16 Контрольная группа

Figure 14 - The content of polyunsaturated fatty acids in meat starlet when used a feed additive Tseofish (g/100 g)

92 3.4.5 Vitamin content of fish meat of valuable species when used in feed NFA Tseofish

Fish is one of the most important sources of protein, fat, micro and macronutrients, vitamins and vitamin-like substances. Vitamin content of different species of fish is far not been studied. It is known that among species of a family, there are differences in the ability to accumulate in body tissues vitamins. Vitamins contained in almost all tissues of fish. From liposoluble - is A, D, E, K, and from water-soluble - almost all B vitamins [113]. During the studies on vitamin content in the meat of rainbow trout and sterlet, as top dressing which use non-traditional feed additive Tseofish found that the feed additive has no negative influence on the content of vitamins in the experimental groups of fish. This is another prerequisite for the use of the feed additive in feed for fish, since the vitamin content of the normalized using fish meat in our body receives various vitamins. It should also be noted that many of the water-soluble and fat-soluble vitamins include part of the enzymatic systems. Many vitamins in our body are converted into coenzymes. Coenzyme - a substance that binds to the enzyme for its greater activation. Enzyme complexes accelerate a variety of chemical reactions in the body. With their help, regulates metabolism and run certain processes, some substances are broken and formed the other [114]. In the study of the experimental groups of fish meat of rainbow trout on the content of vitamins decrease their level in contrast to the control group of fish was observed. Conversely, in some cases, depending on the type of vitamins showed a tendency to increase the levels of vitamins, especially noticeable when feeding fish was 3% and 4% feed additive Tseofish (Table 18). If the content of vitamin A in the control group was 19,3 ± 0,21 g, the 4 experimental group of rainbow trout (in the diet which adds 4% of non-traditional feed additive Tseofish) his figure was 20.78 ± 0,2, m. e. vitamin A in 4 experimental group increased by 1.39% compared with the control group. According to the content of thiamine notable oktloneny from the norm in the experimental groups radzhunoy trout were not observed, their number was within 0.127 ± 0.007mg to 0.128 ± 0.03 mg. The content of riboflavin in the control and experimental groups of rainbow trout were also within normal limits and ranged from 0.105 ± 0.03 mg to 0.107 ± 0.07 mg. The study observed a marked increase in the levels of niacin increasing percentage of the feed additive to the feed Tseofish. The highest rate levels of niacin were observed in the experimental group 4 rainbow trout, in which feed was added 4% of NFA Tseofish,

93

Table 18 - Contents of vitamins in meat of rainbow trout using a feed additive Tseofish Experimental groups Vitamins Control group 1 experimental 2 experimental 3 experimental 4 experimental group group group group Vitamin A (retinol), g 19,3±0,21 20,1±0,3 20,42±0,01 19,87±0,002 20,78±0,2 Vitamin B1 (thiamine) 0,127±0,007 0,127±0,003 0,128±0,001 0,128±0,001 0,128±0,03 mg Vitamin B2 (riboflavin) 0,105±0,03 0,106±0,009 0,106±0,04 0,107±0,06 0,107±0,07 mg Niacin (vitamin B3 or 5,391±0,02 5,412±0,008 5,426±0,01 5,435±0,003 5,478±0,03 PP), mg Vitamin B5 (pantothenic 0,930±0,002 0,934±0,003 0,936±0,006 0,941±0,001 0,946±0,09 acid), mg Vitamin B6 (pyridoxine), 0,406±0,019 0,412±0,01 0,409±0,002 0,424±0,009 0,431±0,17 mg Folic acid (vitamin B9), 12±0,3 12,6±0,1 12,9±0,2 13±0,08 13,3±0,12 g Vitamin B12 4,47±0,17 4,45±0,01 4,49±0,02 4,51±0,003 4,58±0,011 (cyanocobalamin), g Vitamin C (Ascorbic 2,5±0,23 2,4±0,13 2,5±0,17 2,5±0,20 2,5±0,14 Acid) mg

94 at the same time the level of niacin did not exceed physiological limits for rainbow trout. The content of pantothenic acid, cyanocobalamin, folic acid and ascorbic acid in the control and experimental groups of trout also remained normal. Abnormalities in the level of content pyridoxine not observed was significantly increase its content in the experimental groups compared with the control group, the maximum rate reached its peak in the 4experimental group 0,431 ± 0,17 g. In Table 19 shows the results of analysis on sterlet meat content of vitamins for use in the composition of fish feed unconventional feed additive Tseofish. According to the analysis meat content of vitamins has been found that the vitamin composition of meat sterlets not undergone pathological change, i.e. All values were within the physiological range for sturgeon. Noticed a slight increase in the levels of niacin in the experimental groups starlet that probably explained by the fact that the meat is rich in sturgeon this type of vitamin. The maximum amount of niacin reached in the fourth test group starlet, which were fed with the content of 4% NFA Tseofish.

95 Table 19 - Contents of vitamins in meat starlet using the feed additive Tseofish

Experimental groups Control Vitamins 1 experimental 2 experimental 3 experimental 4 experimental group group group group group Vitamin A (retinol), g 11,9±0,21 12,1±0,3 12,3±0,01 12,5±0,002 12,7±0,2 Vitamin B1 (thiamine) mg 0,226±0,007 0,227±0,003 0,229±0,001 0,228±0,001 0,229±0,03 Vitamin B2 (riboflavin) mg 0,38±0,03 0,40±0,009 0,41±0,04 0,40±0,06 0,42±0,07 Niacin (vitamin B3 or PP), 7,86±0,02 7,88±0,008 7,89±0,01 7,91±0,003 7,92±0,03 mg Vitamin B5 (pantothenic 1,662±0,002 1,664±0,003 1,665±0,006 1,666±0,001 1,669±0,09 acid), mg Vitamin B6 (pyridoxine), 0,818±0,019 0,820±0,01 0,821±0,002 0,821±0,009 0,823±0,17 mg Folic acid (vitamin B9), g 25±0,3 26±0,1 25,5±0,2 26±0,08 29,3±0,12 Vitamin B12 3,18±0,17 3,20±0,01 3,17±0,02 3,21±0,003 3,24±0,011 (cyanocobalamin), g

96 3.4.6 Mineral content of fish meat in the application of NFA Tseofish

An important group of substances belonging to the indispensable nutritional factors and influences the quality and nutritional value of meat and its products are minerals. Minerals found in different parts of the fish body in unequal amounts. Minerals found in the ash obtained by burning the meat and other parts and organs of the fish. The greatest amount of mineral elements contained in the bones. Total amounts of minerals in the body of the fish are 4%. In fish quantitatively predominant phosphorus, calcium, potassium, sodium, magnesium, sulfur, and chlorine (macronutrients). The rest of the detected elements - iron, copper, manganese, cobalt, zinc, molybdenum, iodine, bromine, fluorine, etc. In very small amounts (micronutrients). The bulk of calcium and phosphorus in the body of the fish found in the bones, forming their hard skeleton. Sodium, potassium, phosphorus, magnesium, chlorine included in the sarcoplasmic muscle cells, interstitial fluid, blood plasma. Sulfur is included in the composition of proteins. Great physiological importance is the trace elements that are part of a series of important organic compounds. In marine fish meat contains more minerals than meat fresh. An important difference between marine and freshwater fish is the almost complete absence in the meat of freshwater fish of iodine and bromine. Results of the analysis of the mineral composition of valuable species of fish are shown in Tables 20-21. Studies of the mineral composition of meat of rainbow trout and sturgeon have shown that it is rich with essential functionally significant components that make them biologically valuable product. The mineral components as evidenced by the results obtained are not significantly different for the test and control samples of meat, except iron and zinc, which level is somewhat lower in the meat of rainbow trout fed NFA Tseofish. In our opinion, this may be due to high adsorption capacity of the latter. In the study of the meat of rainbow trout and sterlet in the feed, a feed additive which adds Tseofish content of sodium and potassium was in physiological amounts, which allows maintaining the osmotic pressure of body fluids and participating in the formation of a buffer system of tissues and biological fluids. The amount of manganese in the range - 0,11-0,22 g/100 g of meat of rainbow trout in the experimental groups and 0,016-0,027 mg/kg meat sterlets increases glycogen synthesis, and increases the efficiency of absorption of vitamins C and B, which actively influences growth and development of fish. The content of calcium and phosphorus in the experimental groups of valuable fish species also remained normal, significant changes in the direction of decreasing was observed.

97 Table 20 - Mineral content in the meat of rainbow trout (control and test, mg 100 g)

Indicators Control group Experimental group (n=20) Physiological norm units of (n=20) 1% 2% 3% 4% measurement Macronutrients mg/kg К 380±0,7 411,7±0,3 420±0,21 431,8±0,15 451,8±0,23 60-420 Ca 34,5±0,61 32,5±0,16 34±0,17 36,78±0,13 37,45±0,13 17-270 Mg 25±0,77 24,55±0,2 25,9±0,07 26,43±0,51 27,15±1,43 10-170 Na 54,9±0,7 57±0,19 56±0,19 57,5±0,17 58±0,41 30-130 P 209,9±0,68 208,6±0,24 205,5±0,15 213,01±0,23 215±0,121 110-550 Micronutrients mg/kg Fe 0,8±0,07 0,7±0,014 0,6±0,017 0,62±0,003 0,7±0,58 0,3-4,6 Zn 2,1±0,02 1,79±0,002 1,92±0,002 1,81±0,005 1,90±0,82 0,05-0,60 Mn 0,14±0,004 0,14±0,002 0,15±0,002 0,21±0,003 0,22±0,53 0,016-0,044

Table 21 - Mineral content in the meat of sturgeon (control and test, mg 100 g)

Indicators Control group Experimental group (n=20) units of (n=20) 1% 2% 3% 4% Physiological norm measurement Macronutrients mg / kg К 280±0,032 289±0,043 297±0,069 303±0,058 308±0,048 60-420 Ca 44±0,023 47±0,056 53±0,239 58±0,357 61±0,357 17-270 Mg 66±0,035 69±0,092 72±0,245 75,1±0,95 78,9±0,64 10-170 Na 92±0,02 98±0,002 103±0,42 107±0,032 111±0,56 30-130 P 259±0,34 263±0,204 268±0,394 277±0,053 283±0,102 110-550

98 Micronutrients ug / kg Fe 0,7±0,03 0,8±0,06 0,8±0,05 0,9±0,06 0,9±0,05 0,3-4,6 Zn 0,6±0,35 0,6±0,56 0,7±0,78 0,7±0,78 0,7±0,45 0,05-0,60 Mn 0,015±0,75 0,016±0,81 0,020±0,74 0,023±0,72 0,027±0,53 0,016-0,044

99 After analyzing the obtained parameters of the mineral composition of meat of valuable fish species in the feed, a feed additive which adds Tseofish we came to the conclusion that certain trends in the overall reduction of mineral substances in the meat of rainbow trout and sterlet experimental groups were noted.

3.5 Morphological changes of valuable species of fish meat using NFA Tseofish

Using natural minerals as feed additives in animal feed, birds and fish is becoming more widely used. There is a positive impact clinoptilolite containing tuffs on animals, birds and fish in the form of increasing the viability and growth. Noted the positive impact of these minerals detoxification function of the liver, protein, fat and carbohydrate metabolism in the body [102]. Studies have shown that zeolite is a valuable mineral feed supplement of natural origin that promotes quantitative and qualitative indicators of meat productivity of fish. In fish, the experimental group the macroscopic structure of the internal organs had pronounced abnormalities. Macroscopically liver not enlarged, the capsule is smooth, flat surface, brown, normal consistency, moderate hyperemia. Histological examination of fish liver test group the overall plan of the structure body, its lobes and the main structural components retained. From the central vein radial strands depart hepatic beams constructed of polygonal liver cells. The cytoplasm of liver cells are round, located in the center of the cell. Clumps of chromatin in them stained with hematoxylin purple. There are cells with two nuclei. Hepatic beams closely transplanted with sinusoids that drugs have on the appearance of cracks between the strands of the hepatic cells. The nuclei of endothelial sinusoidal elongated, sometimes protrude into the lumen of the capillary. Interlobular bile ducts together with the ramifications of the portal vein and hepatic artery formed between the hepatic lobules triad. Skeletal muscle in fish presented striated muscle tissue. On longitudinal sections of the muscle fibers are visible shell fibers having the form of the contour line. Under the sarcolemma at the periphery of the nucleus fibers are elongated with small clumps of chromatin. The central part is occupied by myofibrils fibers, gives the fiber longitudinal striations, stand-out differently in different fibers. The spaces between the striated muscle fibers filled layers of loose connective tissue, which receives the name of the endomysium (Figure 15). Adding zeolite to the daily diet of fish for 63 days contributed to increase the thickness of the mucosa of the small intestine compared with the control group. The mucosa of the colon in the control group of fish is slightly thicker than in the experimental group and the size of the crypts more mucosa fish experimental group. The ratio of goblet cells to other cells of the mucosa in the experimental group was higher than the control. Fish gut wall as all mammals consists of three layers. However, the fish in the mucosa of the villi are absent, in their place, there are only folds. The mucosa of the small intestine shows a single layer of prismatic epithelium cells, it’s tall and narrow. The cytoplasm of epithelial eosin stained pink. At the apical end clearly visible striated edges. The cell nuclei mainly oval lying closer to the basal end of the cells. Among the prismatic cells often met

100 goblet cells. Their cytoplasm is filled with mucus, and the nucleus is displaced to the basal part of the cell. Number cup shape cells in the experimental group compared with the control group increased.

1 experimental (1% Tseofish) 2 experimental (2% Tseofish)

3 experimental (3% Tseofish) 4 experimental (4% Tseofish)

Control

Figure 15 – Histo cut striated muscle tissue. Experimental group (1,2,3,4 experienced). Stained with hematoxylin-eosin. 10x40

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Figure 16 - Kidneys histo cut. Stained with hematoxylin and eosin x 200

Histologically, the kidney capsule wall Bowman presented two sheets. The outer layer of the capsule is clearly visible, the cell nuclei are elongated. Inner layer of the capsule is difficult to discern because it interweaves with a ball of capillaries grow into the capsule. Tubule epithelial cells have a cubic shape, round shape of the nucleus with distinct clumps of chromatin and relatively large nucleolus. The cytoplasm of cells is cloudy with a dark pink shade. At the apical end of the cells is well expressed corymbose rim. Tubules convolutes pass in a relatively short gusset department. This thinner tube lined with low prismatic epithelium, with oval nuclei. Their cytoplasm is light; corymbose rims at the apical end of the cells do not (Figure 16). Thus, the introduction into the diet of fish fodder additive Non-traditional feed additive Tseofish at 1%, 2%, 3%, 4% by weight of the diet did not cause pathological changes in the liver, muscle and other organs of experimental fishes.

3.6 Study of the content of pesticide residues in feed and fish

3.6.1 Pesticide residues in fish feed when using nontraditional feed additive Tseofish

The aim of this study is to measure the level of pesticide residues present in samples of feed. In studies, we investigated 180 active substances: insecticides (organochlorine – 14, organophosphorous – 21, pyrethroid – 11, carbamate – 5 and others – 6), fungicides (37), herbicides (15) and acaricide. Analyses were carried out in a Polish scientific laboratory that possesses an implemented ISO/IEC 17025:2005 system. For this purpose, a multi-method based on Matrix Solid Phase Dispersion

102 and a gas chromatography technique with a dual detection system (electron capture detector/nitrogen phosphorous detector) were applied. Pesticide residue levels were evaluated in relation to the following: Acceptable Daily Intakes (ADIs), Acute Reference Doses (ARfDs) derived from toxicological studies and Maximum Residue Levels (MRLs) (EC, 2005).

Table 22 - Determined active substances of pesticides

Chemical group Active substance Acaricides (7) Bromopropylate; dicofol; fenazaquin; hexythiazox; propargite; tebufenpyrad; tetradifon Fungicides Azaconazole; azoxystrobin; benalaxyl; bitertanol; (65) boscalid; bromuconazole; bupirimate; captan; carbendazim, chlorothalonil; cymoxanil; cyproconazole; cyprodinil; dichlofluanid; dicloran; difenoconazole; dimethomorph; dimoxystrobin; diniconazole; diphenylamine; epoxiconazole; fenamidone; fenarimol; fenbuconazole; fenchlorphos; fenhexamid; fenpropimorph; fludioxonil; fluquinconazole; flusilazole; flutriafol; folpet; hexaconazole; imibenconazole; iprodione; iprovalicarb; kresoxim-methyl; mepanipyrim; metconazole; metalaxyl; myclobutanil; oxadixyl; paclobutrazol; penconazole; pencycuron; picoxystrobin; prochloraz; procymidone; propiconazole; pyraclostrobin; pyrazophos; pyrimethanil; quinoxyfen; quintozene; tebuconazole; tebufenpyrad; tecnazene; tetraconazole; tolclofos-methyl; tolylfluanid; triadimefon; triadimenol; trifloxystrobin; vinclozolin; zoxamide

Pyrethroids Acetochlor; atrazine; chlorpropham; clomazone; (27) cyanazine; cyprazine; dichlobenil; diflufenican; fluazifop-p-butyl; flurochloridone; lenacil; metamitron; metazachlor; metribuzin; myclobutanil; napropamide; nitrofen; oxyfluorfen; pendimethalin; prometrine; propachlor; propaquizafop; propazine; propham; propyzamide; simazine; trifluralin

Organochlorine Acetamiprid; acrinathrin; aldrine; alpha-cypermethrin; pesticides (81) alpha-endosulfan; a-HCH; azinphos-ethyl; azinphos methyl; beta-cyfluthrin; beta-endosulfan; b-HCH; bifenthrin; bromophos-ethyl; bromophos-methyl; buprofezin; cadusafos; carbaryl; carbofuran; chlorfenvinphos; chlorpyrifos; chlorpyrifos methyl; coumaphos; cyfluthrin; cypermethrin; deltamethrin; diazinon; dieldrin; dimethoate; endosulfan-sulfate;

103 endrin; esfenvalerate; ethion; ethoprophos; fenazaquin; fenchlorphos; fenitrothion; fenpropathrin; fenvalerate; fipronil; formothion; c-HCH (lindane); HCB; heptachlor; heptachlorepoxide; heptenophos; indoxacarb; isofenphos; isofenphos-methyl; lambda-cyhalothrin; malaoxon; malathion; mecarbam; metacriphos; mevinphos; methidathion; methoxychlor (DMDT); o,p0- DDTs; p,p0-DDD; p,p0-DDE; p,p0-DDTs; paraoxon- ethyl; paraoxon-methyl; parathion ethyl; parathion methyl; permethrin; phosalone; phosmet; pirimicarb; pirimiphos; pirimiphos methyl; profenofos; propoxur; pyridaben; pyriproxyfen; tau-fluvalinate; teflubenzuron; tetradifon; tetrachlorvinphos; thiamethoxam; triazophos; zeta-cypermethrin

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Figure 17 – Origin of samples of the examined feed

Figure 18 - Scheme of feed sample preparation

To be certain of the quality of the results when the proposed method is applied to routine analyses, various internal criteria have been established. The first one is a blank extract that eliminates contamination, which may result from the extraction and clean-up processes, instruments or chemicals. One blank sample was processed in each set of experiments. The second one is used to check extraction efficiency. Recoveries at the second concentration level (0,05 mg kg-1) will be accepted if the majority of recoveries are within the 70–120% range. Since 2007 the third Laboratory regularly has been successfully participating in proficiency testing (PT) schemes organized by the European Commission (European Union Reference Laboratories, National Food Institute at Technical University of Denmark Chemisches und Veterinдruntersuchungsamt Stuttgart) (EUPT) and interlaboratory comparisons (IL). In proficiency tests and interlaboratory comparisons the method described in subchapters 2,3 and 2,4 has been used to search for 180 active substances. None of the tests has given unsatisfactory (z-score>3), negatively negative or positively negative results. In 5 interlaboratory comparisons and 6 proficiency tests, in only one case out of 116 the obtained z-score (-2,1) for azoxystrobin has been in the range of the questionable results (Table 2).

Pesticide residues in feed This study involved the examination of 50 feed material samples in total, from two provinces of Kazakhstan (Figure 17). All detected active substances were classified according to chemical classes as chloroorganic insecticides (IC), organophosphorus insecticides (IP), pyrethroid insecticides (IPYR) and fungicides (F) (Tables 22 and 23). Feed samples without pesticide residues and with residues below and above MRLs are presented in Figure 19. For feed, residues of chlorpyrifos methyl, diazinon, malathion, pirimiphos methyl (IP group), aldrine, DDTs (including metabolites), c-HCH (IC group), cypermethrin, deltamethrin (group IPYR) and tebuconazole (group F) were detected in the range of 0,02–0,88 mg kg-1. Chlorpyrifos methyl was found in five samples at a concentration of 0,05– 0,88 mg kg-1, pirimiphos methyl in three samples at concentrations ranging from 0,02 to 0,25 mg kg-1 and malathion in one, at a concentration of 0,08 mg kg-1. No residues of plant protection products exceeded maximum residue levels (MRLs), which for chlorpyrifos is 3,0 mg kg-1, and for pirimiphos methyl and malathion: 5,0 and 8,0 mg kg-1, respectively (EC, 2005). Chlorpyrifos methyl, malathion and pirimiphos methyl, which all belong to the group of insecticides, are active substances in such preparations as follows: Actellic 20 FU, Pro Store 157 UL, Pro Store 420 EC. Actellic 20 FU is used for disinfection of empty storehouses, grain and fodder silos, and the content of pirimiphos methyl in this preparation is 22.5%. Pro Store is used for disinfecting seed and consumption grain, and it contains 15–42% malathion. Plant protection products can be applied at the stage of primary production of plants, as well as during crop storage. Chlorpyrifos is a commonly applied insecticide, used for pest

control in agriculture and industry all over the world (Lemus and Abdelghani, 2000). Chlorpyrifos is efficient in controlling the population of many insects, and it is used as an insecticide in cereal, cotton, fruit, vegetables and nuts. Chlorpyrifos is moderately toxic for humans and can affect the central nervous system, cardiovascular system and respiratory system (Nolan et al., 1984). The risk related to chronic exposure to chlorpyrifos residues for humans, estimated by means of a reference dose (RfD) of cholinesterase (ChE), is low and amounts to 0,03 mg kg bw-1 day-1 with consideration of an uncertainty factor related to higher sensitivity of organisms with not fully-developed protection mechanisms as calculated by USEPA (0.003 mg kg bw-1 day-1) (IRIS, 2007). The Acceptable Daily Intake (ADI) for a person was established at the level of 0–0.01 mg kg bw-1 day-1 by WHO/PCS and FAO/WHO JMPR in 1999. Tian et al. (2005) suggested that chlorpyrifos has teratogenic and toxic effects on the mouse embryo in doses lower than assessed in previous research carried out on rats. The effect of chlorpyrifos on human and animal safety is still a current problem that is being investigated by the European Commission and USEPA (http://www.tga.gov.au). Saeed et al. (2001) have investigated chlorinated pesticide residues and have found that chlorpyrifos methyl is present in most of the wheat or wheat flour samples in Kuwait. Maver et al. (2007) have analyzed organophosphorous pesticide residues in many commodities, including cereals, in Slovenia. Bai et al. (2006) have investigated organophosphorous pesticide residues in market food, including cereals in China, and they have found that organophosphorous residue levels were below MRLs in cereals. In our study, residues of chlorpyrifos methyl were detected below MRLs in 6,25% of samples. Pirimiphos methyl was detected in 3,75% of samples in this study (the active substance of a preparation known under its commercial name of Actellic) and has been shown to inhibit acetylcholinesterase. Literature data have established that, in research on mammals, the level which does not cause any harmful effects (determined as NOAEL) is 0,5 mg kg-1 of body mass per day (mg kg bw-1day-1). In the case of humans, no inhibition of cholinesterase was found (FAO/WHO, 1992). Pirimiphos methyl did not demonstrate carcinogenic effects in research that was carried out in doses up to 300 and 500 mg kg-1 (the highest test dose) and does not demonstrate teratogenic effects in mice at levels of up to 16 mg kg bw-1 day-1. The research also indicates that this compound is rapidly expelled, and so far, no evidence has been found for its bioaccumulation in the organisms of the examined animals. Malathion, detected in 1,2% of samples, is a commonly applied organophosphorous pesticide with a broad spectrum of insecticide effects. Malathion is used to control the populations of sucking and chewing insects, and is classified as slightly toxic. The most toxic of its metabolites is malaoxon – a product of oxidation, which is also responsible for the insecticidal activity of malathion. A strong relationship between malathion toxicity and the amount of protein in the diet of laboratory rats has also been found (Gallo and Lawryk, 1991). In humans, the dose level at which adverse effects were observed, e.g. on the alimentary, neurological, and respiratory systems, was three times higher in

women than in men. Among other organophosphorous insecticides, malathion can have a negative effect on the immunological system of some species of animals at relatively high doses (Galloway and Handy, 2003). The largest share of samples with residues of plant protection chemicals was found for Kostanay (24,4%). Grain from the province of Almaty contained 20.5% of samples with residues. Organochlorine pesticides were determined in five samples (6,25%): DDTs, aldrin and c-HCH. The presence of DDTs was stated in two samples from Kostanay and one from Almaty. The metabolite p,p0-DDT was determined with the highest concentration (from 0,09 to 0,15 mg kg-1), the metabolite o,p0-DDT was detected in a lesser degree (from 0,05 to 0,13 mg kg-1), and o,p0-DDE had the lowest concentration (from 0,03 to 0,06 mg kg-1). The sum of isomers in samples amounted to 0,17, 0,2 and 0,34 mg kg-1, respectively, and exceeded the MRL in every case (0,01 mg kg-1). The accumulation of organochlorine compounds in foods is still a matter of major concern although the use of most organochlorine compounds (IC) has been banned or restricted in most countries, due to the uncertainty related to the adverse effects that their residues may have after a lengthy period of exposure at low doses. Organochlorine pesticides are not readily degradable in the environment and are lipophilic with a tendency to bioaccumulate, so they can be found at high concentrations in fatty foods, including cow milk. Distribution of organochlorine pesticides has been reported by authors in different types of samples (Chen et al., 2007; Guler et al., 2010). This most probably reflects the usage pattern of these compounds, which are highly persistent, effective and cheap. Over 60% of total organochlorine contamination is due to DDTs components. While the usage of DDTs in agriculture has been banned in Kazakhstan since 1983, nothing is known about its illegal use. Another explanation may be input from other countries around the Caspian Sea (Senthil-Kumar et al., 2001). This assumption could be confirmed by measuring DDTs in local species at several points in Asia. Lindane (c-HCH) was determined in 1 RGM-2M sample (Almaty) with a concentration of 0.12 mg kg_1. The World Health Organization classifies lindane as moderately hazardous, and its international trade is restricted and regulated under the Rotterdam Convention on Prior Informed Consent. In 2009, the production and agricultural use of lindane was banned under the Stockholm Convention on persistent organic pollutants (CEC, 2006; US-EPA,2006; POPRC, 2007). Lindane has been used to treat food crops and forestry products, as a treatment for seeds, soil, livestock, and pets. Lindane is a neurotoxin that interferes with GABA neurotransmitter function. In humans, lindane affects the nervous system, liver and kidneys, and may be a carcinogen (ATSDR, 2005; IARC, 1998). Lindane is a persistent organic pollutant: it is relatively long-lived in the environment, is transported across long distances by natural processes like global distillation, and can bioaccumulate in food chains, though it is rapidly eliminated when exposure is discontinued. Aldrin was determined in 1 wheat sample (Almaty) with a concentration of 0,08 mg kg-1. Aldrin was developed as a pesticide to control soil insects. Its use is

now banned in the European Union, but it is still used in developing countries. Although Aldrin is banned in the EU and Kazakhstan, its release into the environment can occur from products or materials which have been treated with it elsewhere. It directly contaminates soils in countries where it is still used as a pesticide. At an international level, Aldrin is the subject of two proposed UN treaties, is banned under the UNECE POPs protocol and proposed for elimination under the UNEP POPs Convention (POPS, 2009). Yentur et al. (2001) reported that quintozene, lindane, DDTs and its metabolites were detected in a few samples of cracked wheat from Turkish markets. Guler et al. (2010) reported that chlordane isomers, methoxychlor, DDTs and its metabolites, aldrin, dieldrin and endrin, c-HCH, heptachlor and lindane were found in samples of wheat from Turkey. Bakore et al. (2004) reported that all of the wheat samples from India were contaminated with various organochlorine pesticide residues of DDTs and its metabolites, c-HCH and its isomers, heptachlor, epoxide and aldrin. Toteja et al. (2006) confirmed that residues of DDTs (59.4% samples) and isomers of c-HCH (78.2%) were detected in samples of wheat in India. Reksa-Naik and Prasad (2006) have stated that endosulfan organochlorine residues in wheat were reported in all market samples in India. Skrbic (2007) has assessed levels of organochlorine and organophosphate pesticides in wheat from Serbia and they found the following: c-HCH; aldrin; dieldrin; endrin ketone and endrin aldehyde. In recent decades, pyrethroids have increasingly replaced organochlorine pesticides due to their relatively lower mammalian toxicity, selective insecticidal activity, and lower environmental persistence than organochlorine pesticides (Saha and Kaviraj, 2008). Although posing a minimal threat to mammals and avian species, pyrethroids are extremely toxic to bees (Lozowicka, 2013) and aquatic organisms, including fish such as the bluegill and lake trout. Cypermethrin is a pyrethroid classified as a moderately toxic chemical (Macedo et al., 2009). In China, cypermethrin is one of the most potent insecticides, widely used in veterinary products to control lice, flies, and ticks on cattle and sheep, as well as in agricultural formulations to control numerous insect pests on fruits, vegetables, and field crops. It poses a great threat to fish and other aquatic organisms (Cheng et al., 2009). In one grain sample – oats (Kostanay), cypermethrin was detected with a concentration of 0,05 mg kg-1 and deltamethrin was detected in wheat (Almaty) with a concentration of 0,02 mg kg-1. In our study, the percentage of samples containing pesticide residues varied between 28,8% in wheat, 20% in rye and 13,3% in oat and barley (Fig. 3). No multi-residue samples were found among the studied samples – The products that most frequently contained residues of the examined compounds included wheat (28,8%), and residues were found the least frequently in oats and barley (13,3% each) (Fig. 3). .

Figure 19 - Pesticide residues in tested grain samples

Table 23 – Levels, frequencies and concentration ranges of pesticide residues and maximum residue levels (MRL) found in tested grain samples. Number of samples MRL LOD (mg kg- Concentrati WHO (The ЕС 1) Active substance With With residues With residues on range World Health (European residues >MRL EU

γ- HCH (OC) 1 1 0 0,01 0,01 0,12 0,005 Malathion (OP) 1 0 1 10 8 0,08 0,01 Pirimiphos 2 0 2 7 5 0,05; 0,25 0,01 methyl (OP) Tebuconazole (F) 1 1 0 0,15 0,2 0,25 0,01

Oat (N = 15) 2 0 Chlorpyrifos 1 0 1 10 3 0,05 0,005 methyl (OP) Cypermethrin 1 0 1 2 2 0,05 0,01

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(IPYR)

Barley (N = 15) Chlorpyrifos 1 0 1 10 3 0,88 0,005 methyl (OP) Diazinon (OP) 1 1 0 0,01 0,01 0,17 0,01

Rye (N = 15) Pirimiphos metyl 1 0 1 7 5 0,02 0,01 (OP) Total 18 7 11 - - - -

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Table 24 - Long term health effects associated with the pesticides detected in the study.

Acetylcholi Endocrine Reproduction/development Pesticides Carcinogen Mutagen nesterase Neurotoxicant disrupter effects inhibitor Aldrin Yes - ? ? х Yes Chlorpyrifos х - х - Yes ? methyl Cypermethrin ? х ? ? х х Deltamethrin ? х Yes ? х Yes DDT sum ? Yes Yes x Yes ? Diazinon х ? ? ? Yes Yes

γ- HCH ? х ? ? - Yes Malathion ? ? ? ? Yes Yes Pirimiphos methyl Tebuconazole ? - - Yes x x YES: Yes, known to cause a problem, x: No, known not to cause a problem, ?: Possibly, status not identified, and –: No data. a EC Directive 1107/2009 (repealing 91/414).

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Table 25 - Health risk estimation for chronic effects associated with average pesticide residue concentrations. Pesticides Commodity RL (mg kg-1) ADI (x10-3 mg Source EDI (x10-3 mg HQ (%) cHI (%) kg bw-1 day-1) kg bw-1 day-1) Aldrin Wheat 0,00666 0,1 JMPR 1994 0,789 789,0 789,0 p,p’-DDT Wheat 0,01222 10,0 JMPR 2000 1,448 14,5 14,5 o,p’-DDT Wheat 0,01000 10,0 JMPR 2000 1,185 11,8 11,8 o,p’-DDE Wheat 0,00756 10,0 JMPR 2000 0,895 9,0 9,0 γ- HCH Wheat 0,00756 5,0 JMPR 2003 0,895 17,9 17,9 Sum HQ 789 + 53.2

Chlorpyrifos Wheat 0,01044 10,0 Dir 05/72 1,237 12,4 methyl Oat 0,00600 10,0 Dir 05/72 0,007 0,1 Barley 0,06333 10,0 Dir 05/72 0,232 2,3 14,8 Diazinon Barley 0,02067 0,2 EFSA 06 0,076 37,9 37,9 Malathion Wheat 0,01156 30,0 EFSA 06 1,369 4,6 4,6 Pirimiphos Wheat 0,01622 4,0 EFSA 05 1,922 48,0 methyl Rye 0,01067 4,0 EFSA 05 0,072 1,8 49,8 Sum HQ 107,1 Cypermethrin Oat 0,01267 20,0 Dir 05/53 0,015 0,1 0,1 Deltamethrin Wheat 0,01022 10,0 Dir 03/5 1,211 12,1 12,1 Tebuconazole Wheat 0,01533 30,0 EFSA 08 1,8172 6,1 6,1 Sum HQ 6,1

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Table 26 - Health risk estimation for acute effects associated with the highest pesticide residue concentrations. -1 -3 -3 Pesticides Commodity HR P (mg kg ) ARfD (x10 mg kg Source ESTI (x10 mg kg αHI (%) bw-1 day-1) bw-1 day-1) Aldrin Wheat 0,08 3,0 EFSA 2007 9,478 315,9 Chlorpyrifos Wheat 0,16 100,0 Dir 05/72 18,956 19,0 methyl Oats 0,05 100,0 Dir 05/72 0,058 0,1 Barley 0,88 100,0 Dir 05/72 3,227 3,2 Cypermethrin Oats 0,05 200,0 Dir 05/53 0,058 0,0 p,p0-DDT Wheat 0,15 Not appl. JMPR 2000 17,771 - o,p0-DDT Wheat 0,13 Not appl. JMPR 2000 15,401 - o,p0-DDE Wheat 0,06 Not appl. JMPR 2000 7,108 - Deltamethrin Wheat 0,02 10,0 Dir 03/5 2,369 23,7 Diazinon Barley 0,17 25,0 EFSA 06 0,623 2,5 γ-HCH Wheat 0,12 60,0 JMPR 2003 14,217 23,7 Malathion Wheat 0,08 30,0 EFSA 06 9,478 31,6 Pirimiphos Wheat 0,25 150,0 EFSA 05 29,618 19,7 methyl Rice 0,02 150,0 EFSA 05 0,135 0,1 Tebuconazole Wheat 0,25 30,0 EFSA 08 21,325 98,7

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3.6.2 Pesticide residues in fish when using in feed nontraditional feed additive Tseofish

Fish farming is one of the most promising and fastest growing industries production agriculture, due to the high fertility of fish, their rapid growth and low cost of their cultivation, as well as the increasing demand for high quality food products [1,2]. Changing environmental conditions in the regions affected by the state of the fishery ponds, leading to contamination of organic and mineral fertilizers, pesticides from treated fields, runoff from livestock farms, industrial and municipal enterprises. In turn, changes in hydrological and hydrochemical parameters, thermal regime, and other individual characteristics of the water may reduce the natural forage base, significantly affect the growth rate and viability of fish, and their ability to withstand the effects of adverse factors, the accumulation in fish of various xenobiotics and toxicants, distribution disease [3]. Currently intensive technology of cultivation of crops, pollution from industrial wastes cause the accumulation of heavy metals in fish, residual quantities of pesticides, herbicides and other toxins [4]. Among persistent organic pollutants that are found in the environment and in food products, a significant group represented by the residues of highly toxic organochlorine pesticides used for decades mainly as plant protection products [5, 6]. Since some of the compounds half-life measured in years, they may accumulate in the water and sediments. As a result, they can penetrate into the aquatic organisms and it is dangerous for the health of consumers of seafood. [7,8,9]. Pesticides belonging to the class of organochlorines (OCs) have been banned worldwide since the early 1980s due to their toxicity, stability, high lipophilicity, long biological half-life, and, therefore, a high degree of bioaccumulation in the food chain [10,11]. Despite that their connection is still found in aquatic organisms. Protection of the environment from the destructive power of human impact at the moment, perhaps the most pressing in the world. One of the main environmental problems is the problem of water conservation, quality condition which affects the interests of many sectors of the economy and social development [12,13, 14]. Results of the study on fish muscle content of residues of organochlorine compounds are shown in Table 27. The data obtained in the course of studies show that were marked by exceeding the maximum permissible concentrations of toxic elements in fish. Noteworthy is a significant difference in the qualitative and quantitative composition of organochlorine pesticides in fish of Lake Balkhash. In samples of fish of Lake Balkhash basis pesticides constitute a group of hexachlorocyclohexane (α-, β-, γ-HCH) and heptachlor. The total content HCH varies from 0.028 to 0.192 g / kg. In this case the individual isomers of HCH is the major γ-HCH, ie it testifies to the recent arrival of hexachlorocyclohexane in the environment. Thus, if we analyze the ratio of

117 isomers in all the samples studied, it can be concluded that Lake Balkhash is exposed now contaminated with pesticides hexachlorocyclohexane group, because the content of γ-isomer of HCH in fish larger than α-HCH. The content of residual amounts of α-HCH exceeds the permitted limit in samples of meat bream, and the content of γ-HCH exceeded the permissible norm once in three species of fish (catfish, perch and bream). The highest content of residual amounts of α-HCH and γ-HCH was detected in bream. Apparently this is due to the fact that these organochlorine pesticides, especially γ-HCH is practically insoluble in water, have a pronounced cumulative properties, the ability to accumulate in body fat and muscle tissue of animals, birds and fish. Hexachlorocyclohexane (hexachloran, HCH, S6N6Sl6). Yad contact and systemic action. There are nine different isomers. The most toxic gamma isomer: technical HCH contains its about 12-25%. BHC used in the form of a 12% dust, 25% of the powder on phosphate flour, 20% mineral oil emulsion. Its use in the timber industry to protect wood against insect infestation and as antiseptic additives thereto. Gamma-HCH toxic hexachlorane 4-10 times. Cumulative properties hexachlorane pronounced: In the body, it lingers for a long time in the adipose tissue. On fish HCH acts as the nerve poison, fish initially nervous, respiratory rhythm accelerated, they tend to jump out of the water, then there are seizures, loss of balance, lack of coordination of the fins, tilting to one side, severe convulsions, increased response to stimulation, decreased respiratory rate and after some time, respiratory paralysis. The reversibility of the poisoning of fish possible when migrating fish in fresh water from the state of overturning. Noted poisoning by eating poisoned fish food organisms [16]. The content of residues of DDT and its metabolites in samples of fish of Lake Balkhash has not been revealed. The concentration of heptachlor in meat walleye varies 0.001 mg / kg, and in other fish species its contents were noted. Heptachlor - organochlorine compound from the group polihlortsiklodienov - a group of drugs, which after application to the soil relatively quickly oxidized, non-systemic insecticide highly toxic contact action, highly resistant to destruction, belongs to the so-called "dirty dozen". Heptachlor is used as a powerful insecticide contact action against insect pests of corn, sunflower sprouts, some crops (sorghum), sugar cane and beet, termites, ants, thrips, weevils, beetles, larvae of May and June beetles, both cultivated so and uncultivated soils. In some cases, it may be applied by fumigation. At concentrations of insecticidal application, it is non-phytotoxic, has no herbicidal properties. Heptachlor not only protects the seedlings from damage by insects, but also stimulates germination. Also used as a seed dressing. In addition, heptachlor - great soil insecticide, but he is persistent in soil and plants and is gradually moving into their epoxide, wherein somewhat greater toxicity to warm-blooded animals than heptachlor itself. Currently, in accordance with the Stockholm Convention of 23 May 2001, has a global ban on the production, use and sale of [17, 18].

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Table 27 - Results of studies on fish pesticides

Name of Acceptable The test fish indicators standards (pesticides), for ND Carp Catfish Pike Bream mg / kg, not more α-HCH 0,03 - 0,0005±0,0 - 0,089±0,13 21 4 β-HCH 0,03 - - - - γ-HCH 0,03 0,028±0,10 0,092±0,08 0,040±0,12 0,097±0,01 (Lindane) 2 7 6 0 DDE 0,3 - - - - DDD 0,3 - - - - DDT 0,3 - - - - Heptachlor Not norm/ - - 0,001 -

Thus, we should talk about the small contamination of Lake Balkhash organochlorine pesticides. Of all the species on the content of these contaminants can be identified bream, catfish and walleye. Our research on the content of pesticide residues in meat of fish showed that their number exceeds the permissible limits on regulatory documentation in some species of fish, especially a significant content of residual amounts of γ-HCH observed in samples of meat bream. The fact that after more than 30 years after the ban of the use of heptachlor and HCH, their residues, although in trace amounts, yet discovered, guards and forced to take urgent measures to prevent the contamination of food and environmental objects.

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4 GENERALIZATION AND EVALUATION OF RESEARCH RESULTS

Analysis of our results showed that in all the markets of feed in the Almaty region discovered mineral feed additives of low quality, which is implemented as a zeolite. This mineral feed additive does not comply with the animal health requirements POE to these supplements are considered dangerous for consumers. The quality and safety of the feed additive is not guaranteed and should therefore be considered the animal health requirements applicable to the production of raw materials for zeolite forage preparation. When studying the chemical composition of nontraditional feed additive Tseofish have been found in samples of zeolites high content (about 56%) of Si/Al ≥5, indicating the presence of a high silica zeolite (clinoptilolite). Also in the feed additive Tseofish have been found significantly higher content of calcium, sodium, manganese, magnesium and phosphorus. In appearance Tseofish feed additive is a fine crystalline powder with a light-colored pinkish tinge, odorless, mechanical impurities with crystal size of not more than 0,05 mm. This food additive has a taste of chalk or clay. When an electron-microscopic study noted complex microstructure of zeolites, their pores are formed microcrystals and aggregates. The results of the study it can be concluded that nontraditional feed additive Tseofish is not toxic at a ratio of 1-4% of the fish, as they are made of completely harmless natural mineral zeolite. This is most likely due to a lack of food, and has no relation to the toxicity of the feed additive. It was found that the introduction of the feed additive Tseofish in fish feed had a positive effect on the growth of rainbow trout and sterlet juvenile and their physiological state. Absolute growth of juveniles for 61 day of growing in the experimental basins with the addition of NFA Tseofish in quantities of 1, 2, 3, 4% in relatiion to the control was higher. Indicators of average daily growth of fish in experimental groups also exceeded the control group in the two species of fish. The physiological state of the two-year-rainbow trout and sterlet, when added to the composition of feed NFA Tseofish in quantities of 1, 2, 3, and 4% at the end of cultivation in line with the norm, as in the experimental groups and the control. From this we can conclude that the feed additive does not cause abnormalities in the physiological status of the fish and it can be used as feed additives in the feed. The physiological state of the two-year-old trout at adding the natural mineral zeolite in the mixed fodder in the amount of 1 and 3% in the end of growing complied with the norm both in the experimental and control groups. As a result of research of fish breeding and biological and hematological parameters of the two-year- old trout which feed was added with 1 % and 3 % natural mineral zeolite we have obtained the following data: the absolute growth of young fish for 61 days of cultivation in the experimental pools with the addition of 1 and 3 % of zeolite relative to the control was equal to 116 and 105 %, respectively. Average daily gain in the first case (1%) exceeded those of the control group and the second experimental group by 0.32 and 0.15. The survival rate in all cases was 100%.

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The hemoglobin content in the blood of rainbow trout in the experimental variants was 9.8±0.36 (1% zeolite) and 9.6 ± 0.39 g/l (3% zeolite), respectively and in the control it was-8.7±0.57 g/l. On other hematological parameters, the number of white blood cells as well as the number of immature red blood cells, there were minor differences. Thus, the introduction of 1 and 3 % of the natural mineral zeolite into the feed of a two-year-old rainbow trout and sterlet did not have a negative impact on fish-breeding and biological and hematological parameters of fish body. According to the obtained results we can conclude about a good physiological condition of two-year-old rainbow trout. The nutritive value of the fish and its physiological role as a source of bioactive substances for humans is dependent on the proportions of proteins, fats and mineral substances, the study of the chemical composition of the fish meat is an important part of the veterinarysanitary examination The addition of various amounts of the additive to the fish feed does not only improve the aesthetics of the outerappearance of fish products but also increases their storage life, as wellas the content of vitamins, mineral elements, and food materials. The results of this study confirm that the zeolites have a positive effect on the chemical composition and physical and biochemical features of the meat. A negative effect of clinoptilolite hasn't been determined. From a practical point of view, the rainbow trout (O.mykiss) fed on RGM-2M with up to 4% zeolite contains a higher amount of essential amino acids desirable in a daily diet. This study shows the increase in the level of the polyunsaturated fatty acids. Moreover, using the zeolites as the feed additive for the fish can be a significant part of a comprehensive program to control the fish meat quality. The introduction of the natural zeolites from the Chankanay deposit into the diet of the fish in the amount of 1–4 % weight of the diet does not cause pathological changes in the liver, muscles and other organs of the fish in the experimental group. Therefore, it has no negative effects on the proteolytic enzyme systems of the fish or on breeding. Our studies show that zeolites are a valuable mineral feed additive of natural origin that promotes the production of the fish meat both qualitatively. An inherent element of cereal protection against diseases, weeds and pests, besides intensification of crop cultivation, changes in agrotechnology and crop structure, is the common use of pesticides. In the case of cereal production, they are used both at the stage of primary production and during storage. The application of plant protection chemicals, although highly necessary and effective, can cause the transfer of hazardous substances into the food chain. Protection of cereals, both in the field and in storage houses, should be carried out in the proper manner, including the pest alert system, preservation of the principles of good plant protection practices, and the amounts of hazardous residues of plant protection products should not exceed the MRL values, which are regarded as not causing any adverse effects on human or animal health. Ensuring health safety for the grain produced in Kazakhstan is an absolute priority in crop production. This study has shown that only a small percentage of the examined cereal originating

121 from the entire area of Kazakhstan (22.5%) contained residues of plant protection chemicals at low concentration levels and poses a minimal threat to human or animal safety. – According to the selected risk assessment parameters (food commodities, consumption, and body weight), detected pesticide residues present a negligible hazard to consumers but further attention should be given. – A long term monitoring program for different areas of Kazakhstan is necessary in order to reach more accurate conclusions. – 8.75% of all samples exceeded MRLs. There is a problem in the case of aldrin (1 wheat sample), DDTs (3 wheat samples), lindane (1 wheat sample) and tebuconazole (wheat). – The most frequently detected pesticide was chlorpyrifos methyl. – The most frequent chemical categories detected among monitored products, were organophosphates, organochlorines and pyrethrins.

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5 CONCLUSION

1. As a part of non-traditional feed additive Tseofish zeolite is 56%; and also contains calcium, sodium, manganese, magnesium and phosphorus. In appearance Tseofish feed additive is a fine crystalline powder brown with pinkish hue, odorless solids with crystal size less than 0.05 mm. This feed additive has the flavor of chalk or clay. Electron-microscopic study notes complex microstructure of zeolites, their pores is formed by micro-crystals and aggregates. 2. When conducting experiments on the toxicity of non-traditional feed additive on the body guppy fish, which was added to the feed feed additive Tseofish at doses of 1%, 2%, 3% and 4% to the basic diet, death of fish in the 1, 2, 3 and 4 experienced groups not mentioned. Thus, alternative feed additive Tseofish is non-toxic, and it is perfectly safe as a feed additive for fish. 3. During the research it was found that the introduction of the feed feed additive Tseofish had a positive impact on the growth of juvenile rainbow trout and sturgeon and their physiological state. Absolute growth of fry rearing for 61 hours in the experimental pools at NFA Tseofish additive in quantities of 1, 2, 3, and 4% relative to the control was higher. Average daily gain in fish groups also experienced higher than the control group in the two species. 4. Physiological state of rainbow trout yearlings and starlet when added to the composition of feed NFA Tseofish in quantities of 1, 2, 3, and 4% at the end of cultivation conform to the standards in the experimental and control groups. 5. As a result of sensory studies revealed that the muscles of fish experimental groups was firm, supple, elastic, with pressure on the skin of a finger fossa does not remain, the smell - specific, fresh. When cooking broth sample was transparent, fragrant. The pH of the meat of rainbow trout and sturgeon experimental groups were normal and varied from 6.6 to 6.8. When setting reaction copper sulphate reaction was positive. When microscopy was noticeable decrease in the number of microbes, depending on the concentrations of NFA Tseofish. In the fourth test group of fish (4% Tseofish) was observed on the surface of bacteria 2-3 rainbow trout, 7-8 bacteria in sterlet, in the deep layers of bacteria were observed. Indicators amino ammoniacal nitrogen was normal and was, on average, in the experimental group 1,24 ± 0,25 mg, while the control group - 1,25 ± 0,2 mg. When cooking broth samples potsnovke clear, with a specific smell of fish. Peroxidase reaction was positive when the reaction Nessler filtrate was transparent and haze yellowing was observed. 6. When added to the diet Tseofish feed additive in the meat of rainbow trout marked increase in the number of proteins compared to the control was observed a slight increase in moisture content of the meat the first and second experimental groups, which increased to 0.23%, the third experimental group - 0.1 %, the fourth - 0.2%. The fat content in the fish meat experimental groups increased by 0.37%, 0.18%, 0.55, 0.74%, respectively, compared with the control group. The ash content in the two groups at almost the same level (in the control group and 1.43 in the first test group, 1.43 g / 100 g). Only in the third and fourth group, showed a slight increase in the amount of ash 0.27%. Digestibility of the feed in the

123 experimental groups of rainbow trout remained normal when making non- traditional feed additive Tseofish (within 97,65-98,67%). Also noted the increase in the content of proteins, fats in meat starlet in the application of non-traditional feed additive Tseofish. If the meat samples in the experimental group 1 (1% of the diet fodder additive Tseofish) is equal to the amount of protein was 19.7%, while in the third and the fourth experimental group is significantly increased to 20.3% and 20.9% respectively. If the control group equal to the amount of fat was 19.4%, while in the second and third groups is significantly increased to 20.3% and 21.6%. The moisture content and ash in the experimental and control groups did not change. Digestibility of the feed additive sturgeon also remained within normal limits, as in the experimental groups of rainbow trout. 7. Under experimental conditions found that pesticide residues contain 28.8% of the samples in the feed sample without additive, adding the feed additive of 2% - 20%, 3% and 4 feed additive - 13% sample of the feed. Among the investigated samples of feed multi-residue samples were found.

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6 PRACTICAL PROPOSAL 1 We established veterinary and sanitary requirements for the production and technology of zeolites Chankanayskogo field for feed production allow us to recommend a new feed additive "Tseofish." 2 Veterinary-sanitary examination of fish by using different doses of the feed additive Tseofish (safety, chemical composition of the organoleptic and physico-chemical parameters, biological value of meat) can be used in an amount of 1,2,3,4 Tseofish% of the basic diet of fish. 3 The ability of the feed additive Tseofish raise safety, productivity, and improve complex organoleptic characteristics, as well as the chemical composition and biological value of fish can recommend it for fish farming.

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7 REFERENCES

1 Постановление Правительства Республики Казахстан. О Концепции развития рыбного хозяйства в Республике Казахстан на 2007-2015 годы: утв. 6 октября 2006 года, №963. 2 Суслова И.А., Острякова А.Г. Влияние углеводного концентрата на продуктивность и качество молока коров// Молочнохозяйственный вестник. – 2011. - №2. – С.17. 3 Ермухаметова Ж.Ж. Ветеринарно-санитарная оценка при гельминтозах в Павлодарской области: автореф. … канд. вет. наук: 16.00.06. – Алматы: КазНАУ, 2009. – 3 с. 4 Сарсембаева Н.Б., Уразбекова Д.С., Джусупбекова Н.М., Жанабилова Ж.М. Применение новой кормовой добавки «Цеофиш» созданной на основе НКД «Цеос» и рыбной муки// Исследования,результаты. – 2008.- № 1. - С.99- 101. 5 Коломбет Л.В., Бикетова Д.Х. Тихие враги // Жизнь без опасностей. - 2009. - №3.- С. 45-50. 6 Тейлор-Пикард Д. Скрытое влияние микотоксинов на родительское стадо // Птица и продукты. - 2011. - № 1. - С. 36-39. 7 Гулюшин С., Зернов Р. Комплексный подход к профилактике микотоксикозов // Птицеводство. - 2011. - № 5. - С. 15-17. 8 Карболин П.В. Использование в рационах цыплят-бройлеров глауконита и цеолита: автореф. … канд. с/х. наук: 06.02.08. – Курган: Курганская государственная сельскохозяйственная академия имени Т.С. Мальцева, 2011. – 3 с. 9 Russell R., Paterson M. How will climate change affect mycotoxins in food? //Food research international. - 2010. - V. 43. - P. 1902-1914. 10 Zain M.E. Impact of mycotoxins on humans and animals // Journal of Saudi Chemical Society. - 2010. - V. 15. - P. 129-144. 11 Сарсембаева Н.Б., Уразбекова Д.С., Джусупбекова Н.М., Жанабилова Ж.М. Физико-химические свойства НКД «Цеос», «Шунгистим», «Бентос», «Кудюр-Ю», «Кудюр-Б» // Исследования, результаты.- 2008. - №2. - С.97-99. 12 Кузнецов А.А. Рыбоводно-биологическая эффективность применения природного цеолита-клиноптилолита в составе комбикормов для радужной форели и сибирского осетра: автореф. … канд. биол. наук: 03.00.32. – М.: Московская государственная технологическая академия, 2002. – 3 с. 13 Крюков В.И. Рыбоводство. Интенсификация прудового рыбоводства. – Орел: ОрелГАУ, 2006. – 3 с. 14 Латыпова Г.Ф. Использование природных минеральных добавок для повышения биоресурсной продуктивности кур: автореф. … канд. биол. наук: 03.00.32. – Уфа: Башкирский государственный аграрный университет, 2006. – 3 с. 15 Karamanlis X., Fortomaris P., Arsenos G., Dosis I., Papaioannou D., Batzious C., Kamarianos A. The effect of a natural zeolite (clinoptilolite) on the

126 performance of broiler chickens and the quality of their litter // Asian-Australian Journal of Animal Science. – 2008. - №11. – P.1642-1650. 16 Постановление Правительства Республики Казахстан. Об утверждении Технического регламента "Требования к безопасности кормов и кормовых добавок": утв. от 18 марта 2008 года, №263. 17 Yildirim O., Turker A., Senel B. Effects of natural zeolite (clinoptilolite) levels in fish diet on water quality, growth performance and nutrient utilization of tilapia (Tilapia zillii) fry // Fresenius Environmental Bulletin. – 2009. - №9. – P. 1567-1571. 18 Цветкова А.М. Эффективность использования сорбентов в кормлении молодняка кроликов: автореф. … канд. с/х. наук: 06.02.08. – Родники: ГНУ Научно-исследовательский институт пушного звероводства и кролиководства имени В.А. Афанасьева, 2013. – 3 с. 19 Боровков М.Ф., Фролов В.П., Серко С.А. Ветеринарно-санитарная экспертиза с основами технологии и стандартизации продуктов животноводства. - Санкт Петербург: Лань, 2007. – 6 -19 с. 20 Adeyemi D., Ukpo G., Anyakora Ch., Unyimadu J. Organochlorine Pesticide Residues in Fish Samples from Lagos Lagoon, Nigeria // American Journal of Environmental Sciences. – 2008. - №6. – P.649-653. 21 Khalaf Allah S. Effect of pesticide water pollution on some haematological, biochemical and immunological parameters in Tilapia nilotica fish // DTW. Deutsche Tierarztliche Wochenschrift. – 1999. - №2. – P.67-71. 22 Елемесов К.Е., Шуклин Н.Ф. Ветеринарно-санитарная экспертиза, стандартизация и сертификация продуктов. – Алматы: Кредо, 2002. – 201 c. 23 Горегляд Х.С., Макаров В.А. Ветеринарно-санитарная экспертиза с основами технологии продуктов животноводства. - М.: Колос, 1981.- 4 с. 24 Макаров В.А. Ветсанэкспертиза пищевых продуктов на рынках и в хозяйствах. - М.: Колос, 1992. - 5 с. 25 Макаров В.А., Фролов В.П., Шуклин Н.Ф. Ветеринарно-санитарная экспертиза с основами технологии и стандартизации продуктов животноводства. - М.: БО Агропромиздат, 1991. – 4 с. 26 Шуклин Н.Ф., Кирикбаев С.К., Махышев Т.А. Очерки из истории отечественной ветсанэкспертизы. - Алматы: Кредо, 1999. – 7 c. 27 Давыдов О.Н., Абрамов А.В., Темниханов Ю.Д. Ветеринарно- санитарный контроль пищевых продуктов. – Черкассы: АНТ, 2007. – 5 c. 28 Лоскутов И.Ю. http://www.zakon.kz/60525-veterinarno-sanitarnaja- jekspertiza.html, 13 июня 2008 г. 29 On Approval of Regulatory Legal Acts in the Field of Veterinary. Resolution of the Governments of the Republic of Kazakhstan: `dat. 28 April 2003, №407 30 Law of the Republic of Kazakhstan. On Veterinary: dat. 10 July 2002, №339 31 Маловастый К. Диагностика болезней и ветсанэкспертиза рыбы. – Санкт-Петербург: Лань, 2013. – 237-251 с.

127

32 Береза И.Г. Пособие по ветсанэкспертизе мяса и мясопродуктов при инфекционных, инвазионных и незаразных болезнях животных, птицы и рыбы. - Львов, Подолье, 1994. – 91-105 с. 33 Аганин А.В., Береза И. Г., Бойков Ю. И., Боровков М. Ф., Бурков В. И. Ветеринарно-санитарная экспертиза, стандартизация и сертификация продуктов. – М.: КомСнаб, 2005. – 123-136 с. 34 Житенко П.В., Боровков М.Ф. Ветеринарно-санитарная экспертиза продуктов животноводства. Справочник. М.: «Колос», 2000. – 234-236 с. 35 Васильев Д.А., Мерчина С.В. Лабораторный практикум по ветеринарно-санитарной экспертизе рыбы. – Ульяновск: УГСХА, 2006. – 6-14 с. 36 Грищенко Л.И., Акбаев М.Ш., Васильков Г.В. Болезни рыб и основы рыбоводства. – М.: Колос, 1999. – 456 с. 37 Шуклин Н.Ф., Кирикбаев С.К., Махышев Т.А. Очерки из истории отечественной ветсанэкспертизы. – Алматы.: Кредо, 1999. – 231 с. 38 Правила ветеринарно-санитарной экспертизы морских рыб и икры: утв. 13 октября 2009 г. №462. http://www.mcx.ru/documents/document 39 Правила ветеринарно-санитарной экспертизы пресноводной рыбы и раков: утв. июль 2011 г. http://www.bestpravo.ru/sssr/eh-akty/w9v.htm 40 Постановление Министерства сельского хозяйства и продовольствия Республики Беларусь. О порядке проведения ветеринарно-санитарной экспертизы рыбы и рыбной продукции: утв. 27 апреля 2004 года, №30. 41 FAO, Food and agriculture organization of the United States. Fish is food for the brain as well as good protein. FAO USA, 2000 http://www.fao.org/focus/e/fisheries/nutr.htm 42 Arannilewa S.T., Salawu S.O., Sorungbe A.A., Ola-Salawu B.B. Effect of frozen period on the chemical, microbiological and sensory quality of frozen tilapia fish (Sarotherodun galiaenus) // African Journal of Biotechnology. – 2005. - №4. – P. 852-855. 43 Andrew A.E. Fish processing technology. – Nigeria: University of Llorin press, 2001. – 7-8 pp. 44 FAO, Fisheries and Aquaculture Department, Proteins from fish and fish products. FAO, 2014 http://www.fao.org/fishery/topic/14869/en. 45 Timirkhanov S., Chaikin B., Makhambetova Z., Thorpe A., Anrooy, R. Fisheries and Aquaculture in Kazakhstan: The Current Status. http://www.thefishsite.com/articles/984/fisheries-and-aquaculture-in-kazakhstan- the-current-status19.09.2010. 46 Timirkhanov S., Chaikin B., Makhambetova Z., Thorpe A., Anrooy R. Fisheries and Aquaculture in Kazakhstan: Introsuction. http://www.thefishsite.com/articles/984/fisheries-and-aquaculture-in-kazakhstan- the-current-status10.09.2010. 47 Решетников Ю.С. Атлас пресноводных рыб России. – М.: Наука, 2002. - 379 с. 48 Дорофеева Т.А. Рост и биологические особенности радужной форели при использовании ферментных препаратов и антиоксидантной смеси: дис.

128

…канд.с/х наук: 06.02.04. – Владикавказ: Горс. гос. Аграрный университет, 2009. – 54 с. – Инв. №04201052985. 49 Назарова М.А. Липидный состав тканей радужной форели Parasalmo Mykiss (Walbaum, 1792) выращенной на различных комбикормах: дис. …канд.биол.наук: 03.02.06. – Петрозаводск: Институт биологии Карельского научного центра, 2014. – 11 с. 50 Emre Y., Okumus I., Maltas O. Trout farming. Marine aquaqulrure in Turkey // Turkish Marine Reserch Faundation, 2007. - №2. P. 21–26. 51 Behnke R.J.; Tomelleri J.R. "Rainbow and Redband Trout". Trout and Salmon of North America // New York: The Free Press. p. 67. ISBN 0-7432-2220-2. 52 Tacon A.J., Metian M. Global overview on the use of fish meal and fish oil in industrially compounded aquafeeds: trends and future prospects // Aquaculture, 2008. - №285. P.146–158. 53 Hardy R.W., Barrows F.T. Diet formulation and manufacturing. - New York: Academic Press Inc., 2000. - 505-600 pp. 54 Chalamaiah M., Balaswamy K., Narsing Rao G., Prabhakara Rao P., Jyothirmayi T. Chemical composition and functional properties of mrigal (Cirrhinus mrigala) egg protein concentrates and their application in pasta // Journal of Food Science and Technology, 2011. - №3. – P. 514-520. 55 Pachanawa A., Phumkhachorn P., Rattanachaikunsopon P. Potential of Psidium guajava supplemented fish diets in controlling Aeromonas hydrophila infection in tilapia (Oreochromis niloticus) // J. Biosci. Bioeng., 2008.- № 106. - P. 419-424. 56 Glencross B.D. Pilot assessment of canola meal and oils as aquaculture feed ingredients // Final Report to the Grains Research and Development Corporation, 2003. pp 124 57 Glencross B.D., Hawkins W.E., Evans D., McCafferty P., Dods K., Maas R., Sipsas S. Evaluation of prototype lupin protein concentrates for use in nutrient dense aquaculture diets when fed to rainbow trout (Oncorhynchus mykiss) // In: 11th International Society for Nutrition and Feeding of Fish Meeting, 2004. 2nd – 7th May, Phuket, Thailand. 58 Smith D.M., Tabrett S.J., Irvin S.J., Barclay M.C. Lupins - an alternative protein source for use in shrimp feeds // VIII International Symposium on Aquaculture Nutrition (VIII SINA), Nov 2006, Mazatlan, Mexico. 59 FAO The State Of World Fisheries and Aquaculture Rome, 1998. 60 FAO Fisheries Report, Rome, 2000. No. 673. 61 FAO State of World Fisheries and Aquaculture Rome, 2007. 62 Iyayi, E. Enzyme supplementation of feeds for improved quality and utilization. The Guardian Agrocare Section, 2003. Sunday 23rd March, pg.54. 63 Federation of Scottish Aquaculture Producers. Scottish Aquaculture “A Vision for the Future”. Meeting with the Scottish Aquaculture Minister 15 January 2002 Pentland House, Edinburgh 64 Huntington T., Frid C., Banks R., Scott C., Paramor O. Assessment of the Sustainability of Feed fisheries Producing Fish Meal and Fish Oil’//Report to RSPB. Poseidon Aquatic Resource Management Ltd, Lymington, Hampshire,2002. P. 62.

129

65 Hutchings J. A. Collapse and recovery of marine fishes//Nature, 2000. №406 P. 882-885 66 Caballero M. J., Obach A., Rosenlund G., Montero D., Gisvold M., Izquierdo M. S. Impact of different lipid sources on growth. Lipid digestibility tissue fatty acid composition and histology of rainbow trout. Oncorhynchus mykiss // Aquaculture, 2002. - №214. P. 253–271. 67 Ackman R.G. Nutritional composition of fats in seafoods // Progress in Food and Nutrition Science, 2000. №13. P.161–241. 68 Caballero M. J., Obach A., Rosenlund G., Montero D., Gisvold M., Izquierdo M. S. Impact of different lipid sources on growth. Lipid digestibility tissue fatty acid composition and histology of rainbow trout. Oncorhynchus mykiss // Aquaculture, 2002. - №214. P. 253–271. 69 Domaizon D. C., Debroas D., Bourdier G. Influence of zooplankton and phytoplankton on the fatty acid composition of digesta and tissue lipids of silver carp: mesocosm experiment // Journal of Fish Biology, 2000. - №57. P. 417–432. 70 Vladau V.V., Bud I., Stefan R. Nutritive value of fish meat comparative to some animals meat // Bulletin UASVM Animal Science and Biotechnologies, 2008. -№65. P. 301–305. 71 Domaizon D. C., Debroas D., Bourdier G. Influence of zooplankton and phytoplankton on the fatty acid composition of digesta and tissue lipids of silver carp: mesocosm experiment // Journal of Fish Biology, 2000. - №57. P. 417–432. 72 Vladau V.V., Bud I., Stefan R. Nutritive value of fish meat comparative to some animals meat // Bulletin UASVM Animal Science and Biotechnologies, 2008. -№65. P. 301–305. 73 Trbović D., Vranić D., Đinović J., Borović B., Spirić D., Babić J., Spirić A. Masnokiselinski sastav i sadržaj holesterola u mišićnom tkivu jednogodišnjeg šarana (Cyprinus carpio) u fazi uzgoja // Tehnologija mesa, 2009. -№50. P. 276–286. 74 Van Bekkum H., Flanigen E.M., Jansen J.C. Introduction to zeolite science and practice. Amsterdam: Elsevier Science Publisher, 2001. 75 Colella C. Application of natural zeolites. Handbook of porous solids 2002; 2:1156–89 76 Colic M., Pavelic´ K. Molekular mechanizmus of anticancer activity of some natural dietetic products. J Mol Med 2000; 78:333–6. 77 Zeo agronomic technolofgy. Feed additive. 2004; http://www.zeoinc.com. Animal feeding. 1999; http://www.atzzeoliti.it/applications.htm. Rotamin Feed Additive Reduction of the digestion system illnesses. 2004; http://www.rotamadencilik.com/rotamin.html. 78 Rimar J., Gavalova Z. Vyuzitie mravcˇanu vapenateho ako prısadydo krmnych zmesı, ale aj ako konzervacnych prıpravkov presilazovanie objemovych krmıv. Vyskumna sprava. Chemko Strazske: 2002. 79 Bindas L., Vajda V., Maskalova I. Vyuzitie acidifikujucich latok a zeolitov v chove osıpanych. V. Dni vyzivy a veterinarnej dietetiky. Zbornık z mezdinarodnej vedeckej konferencie, Hradok: 2002:188–92.

130

80 Skalicka M., Korenekova B., Makoova Z., Nad P. Vplyv aditıv navybrane produkc ne ukazovatele hydiny. V. Dni vyzivy a veterinarnej dietetiky. Zbornık z mezdinarodnej vedeckej konferencie, Hradok: 2002: 237–9. 81 Konakova A. Overenie prıpravku ZeoFeed na parametre uzitkovostivo vykrme brojlerovych kurciat Bratislava: Ustredny kontrolny askusobny u stav polnohospodarsky. 2003: protocol 316. 82 Konakova A. Overenie prıpravku ZeoFeed na parametre uzitkovostinosnı´c- biologicka skuska. Bratislava: Ustredny kontrolny asku sobny u stav polnohospodarsky. 2003: protokol 317. 83 Cincotti A., Lai N., Orrù R., Cao G. Sardinian natural clinoptilolites for heavy metals and ammonium removal: experimental and modeling // Chemical Engineering Journal. 2001. № 84. P. 275-282. 84 Abusafa A., Yucel H. Removal of 137Cs from aqueous solutions using different cationic forms of a natural zeolite: clinoptilolite // Separation and Purification Technology. 2002. Vol. 28, № 2. P. 103-116. 85 Малимин Р.Е. Биокремний органические пористые сорбенты в профилактике акушерской патологии у коров: автореф. … дисс. канд. вет. наук:.16.00.07. – Львов.: ДАВМ, 2000. 20 с. 86 Ярован Н.И. Влияние цеолитов на процессы адаптации у коров // Доклады РАСХН. – Москва, 2008. 43-45 с. 87 Гайдаш А.А., Цуканов В.В. Протекторное влияние цеолитового энтеросорбента в условиях фтористой интоксикации // Экспериментальная и клиническая гастроэнтерология. 2002. № 2. С. 92–95. 88 Голохваст К.С., Паничев А.М. О протекторном действии цеолитов на систему местного иммунитета дыхательных путей / Вестник новых медицинских технологий, 2008а. Т. XV, № 2. С. 217-218. 89 Засекин Д.А. Мониторинг тяжелых металлов во внешней среде и способы их снижения в организме животных: автореф. дисс. д-ра вет. наук. Киев, 2002. 40 с. (укр.). 90 Албегова Н.Р. Физиологический анализ влияния цеолита ирлит-1 на почечные эффекты хлорида кобальта, его распределение и выведение из организма: дисс. ... канд. биол. наук. Владикавказ, 2004. 223 с. 91 Гайдаш А.А. Структура миокарда, легких, печени, почек и физико- химические свойства соединительной ткани под влиянием фтора и природного цеолита (экспериментальное исследование): автореф. дисс. ... д-ра биол. наук. Новосибирск, 2005. 30 с. 92 Гайдаш А.А., Николаев В.Г., Габуда С.П. Структура миокарда, печени, почек и физико-химические свойства соединительной ткани при воздействии фтора и природных цеолитов // Атлас электронно-микроскопических фотографий, спектров ЯМР и комбинационного рассеяния / Ред. Л.Д. Зыкова, С.Г. Козлова. Красноярск, 2005. 110 с. 93 Брин В.Б., Бузоева М.Р., Гаглоева Э.М. Профилактика накопления и стимуляция экскреции тяжелых металлов с помощью применения цеолитоподобных глин «Ирлитов» в эксперименте // Вестник новых медицинских технологий. 2006. Т. XIII, № 3. С. 44-45.

131

94 Брин В.Б., Бузоева М.Р., Гаглоева Э.М. Профилактика проявлений токсической нефропатии вызванной тяжелыми металлами (хлоридом кобальта, хлоридом ртути), с помощью применения цеолитоподобных глин Ирлитов // Вестник новых медицинских технологий, 2007. Т. XIV, № 1. С. 196- 198. 95 Шуклин С.И. Использование хотынецких цеолитов для коррекции морфофункционального состояния яичников у свиней при хронической интоксикации соединениями свинца: дисс. ... канд. биол. наук. Курск, 2006. 151 с. 96 Бузоева М.Р. Экспериментальный анализ профилактического влияния цеолитоподобных глин ирлитов на почечные эффекты хлорида кобальта, его распределение и выведение из организма: автореф. дисс. канд. мед. наук, Владикавказ, 2008. 22 с. 97 Гаглоева Э.М. Влияние энтерального введения природного минерала РСО-Алания Ирлит-1 на почечные эффекты хлорида ртути, распределение и динамику выведения ртути из организма в эксперименте: автореф. дисс. канд. мед. наук. Владикавказ, 2008. 22 с. 98 Папуниди Э.К. Экспериментальное обоснование разработки средств профилактики при сочетанном воздействии на животных токсичных элементов, микотоксинов и пиретроидов автореф. дисс. д-ра. биол. наук. Казань, 2008а. 38 с. 99 Ivkovic S., Zabcic D. The effect of tribomechanically activated zeolite (TMAZ) on total antioxidant status of healthy individuals and patients with malignant disease // Free Radical Biology and Medicine. 2002. № 33 (Suppl. 1). P. 100-102. 100 Zarkovic N., Zarkovic K., Kralj M. et al. Anticancer and antioxidative effects of micronized zeolite clinoptilolite // Anticancer Res. 2003. № 23 (2B). P. 1589-1595. 101 Соловьев С.В., Соловьев В.Г., Гагаро М.А. и др. К механизму защитного эффекта цеолитов при стресс-воздействии // Материалы международного эмбриологического симпозиума «Югра-эмбрио. Закономерности эмбриофетальных морфогенезов у человека и позвоночных животных», 21-22 октября 2004 г. Ханты-Мансийск: Издательский центр ХМГМИ, 2004. С. 340-342. 102 Sverko V. et al. Natural micronized and clinoptilolite mixed with extract Urtica dicia L. as possible antioxidant // Food Technol. Biotechnol/ 2004. Vol. 42. P. 189-192. 103 Колотилова М.Л., Иванов Л.Н. Цеолитсодержащий трепел (ЦТ) в экспериментальной терапии язвы желудка у подопытных кроликов // Российский журнал гастроэнтерологии, гепатологии, колопроктологии. 2006. № 5. Ст. 418. С. 112. 104 Oguz H., Kececi T., Birdane Y.O., Onder F., Kurtoglu V. Effect of clinoptilolite on serum biochemical and hematological characters of broiler chickens during aflatoxicosis // Res. Vet. Sci. 2000. № 69. Р. 89-93.

132

105 Федорова А.И. Структурно-функциональная характеристика лимфатических узлов при применении фитоминерального комплекса в нормальных условиях гемо- и лимфоциркуляции и при экзо- и эндотоксикозе: автореф. дисс. ... канд. мед. наук. Новосибирск, 2000. 106 Huwig A. et al. Mycotoxin detoxication of animal feed by different adsorbents // Toxicology Letters. 2001. № 122. P. 179-188. 107 Tomasevic-Canovic M. et al. The effect of exchangeable cations in clinoptilolite and montmorillonite on the adsorption of aflatoxin B1 // J. Serb. Chem. Soc. 2001. Vol. 66, № 8. P. 555-561. 108 Богомолов Н.И., Богомолова Н.Н., Болтян В.А. и др. Шивыртуйские цеолиты на службе здоровья животных и человека // Чита: Экспресс- издательство, 2005. 148 с. 109 Ivkovic S., Baranek T., Bendzko P., Schulz J. TMAZ nanoparticles as potential drugs influencing the cellular signal transduction pathways // Nanotech. 2005. Vol. 1, Chapter 2: Medical Applications. P. 85-88. 110 Колотилова М.Л., Иванов Л.Н. Цеолитсодержащий трепел в экспериментальной гепатологии // Патологическая физиология и экспериментальная терапия. 2005. № 3. С. 12-13. 111 Колотилова М.Л., Иванов Л.Н. Экспериментальная терапия острого токсического гепатита у кроликов цеолитсодержащим трепелом // Российский журнал гастроэнтерологии, гепатологии, колопроктологии. 2006. № 5. Ст. 290. С. 79. 112 Ortatatlia M., Oguzb H., Hatipoglua F., Karaman M. Evaluation of pathological changes in broilers during chronic aflatoxin (50 and 100 ppb) and clinoptilolite exposure // Research in Veterinary Science. 2005. № 78. Р. 61-68. 113 Попп Е.А., Правоторов Г.В., Новиков В.Д., Склянов Ю.И. Морфологическое исследование плаценты и печени беременных крыс и их плодов при экспериментальном эндотоксикозе и протекции цеолитами // Морфология. 2005. № 4. С. 47-50. 114 Горьковенко Н.Е., Макаров Ю.А., Квартников А.В., Серебрякова В.А. Применение цеолитов для детоксикации бройлеров // Птицеводство. 2006. № 5. С. 18-19. 115 Bereczk I. Production of a zeolitic mineral composition for binding radioisotopes and heavy metal ions, 1998. Hungary Pat. Teljes HU 77955 A2, 1-5. 116 Харьковский А.В., Третьякова Т.А. Влияние природных цеолитов на выведение цезия-137 и стронция-90 из организма мышей // Бюл. СО РАМН, 1998. № 3. С. 122-124. 117 Рачиков С.В. Изменение содержания микроэлементов и выведение радионуклидов из органов и тканей помесного молодняка крупного рогатого скота при скармливании добавок цеолита: дисс. канд. биол. наук. Брянск, 1999. 118 Красноперова А.П., Лонин А.Ю., Юхно Г.Д., Лебедева Л.Т., Далецкая Н.В. Сорбционно-селективные свойства природного цеолита – клиноптилолита в отношении радионуклидов 90Sr и 137Cs / Вестник

133

Харьковского Национального Университета. 2001. № 532, Серия Химия. № 7(30).С. 143-148. 119 Кривова Н.А., Лаптева Т.А., Селиванова Т.И. и др. Влияние длительного потребления с пищей цеолитов на выживаемость и компенсаторные реакции кишечника у мышей разного возраста после облучения // Радиационная биология. Радиоэкология. 2001. № 41(2). С. 157-64. 120 Бгатова Н.П., Голохваст К.С., Бгатов А.В. и др. Модулирующее действие природного цеолита на структуру пейеровых бляшек в условиях накопления цезия // Тихоокеанский медицинский журнал, 2009. № 3. 74-77. 121 Засекин Д.А. Мониторинг тяжелых металлов во внешней среде и способы их снижения в организме животных: автореф. дисс. д-ра вет. наук. Киев, 2002. 40 с. (укр.). 122 Conception-Rosabal B., Rodriguez-Fuents G., Simon-Carballo R. Development and featuring of the zeolitic active principle FZ: A glucose adsorbent // Zeolites. 1997. № 19. Р. 47-50. 123 Concepción-Rosabal B., Bogdanchikova N., De la Rosa I., Rodríguez- Fuentes G. et al. Bactericidal action of Cuban natural clinoptilolite containing clusters and nanoparticles of silver. Book of abstracts 7th International Conference on the Occurrence, Properties, and Utilization of Natural Zeolites «Zeolite ’06»,16– 21 July 2006, Socorro, New Mexico, USA. P. 88-90. 124 Курамшина Н.Г., Маннапова Р.Т., Топурия Г.М., Маннапов А.Г. Южноуральские цеолиты – экобезопасность и влияние на организм птицы, сельскохозяйственных животных. Уфа-Оренбург-Москва: Изд-во БашГАУ, 2007. 248 с. 125 Зедгенизова С.Н., Просекина О.В. Морфологические показатели двенадцатиперстной кишки кур-несушек при использовании якутских цеолитов в качестве кормовой добавки // Аграрный вестник Урала, 2008. - №1 С.41. 126 Лумбунов С.Г., Лузбаев К.В., Александрова Е.А. Природные минералы в животноводстве Бурятии // Химия и компьютерное моделирование. Бутлеровские сообщения. 2006. № 1М. С. 322-323 127 Хаустов В.Н. Витамин К4 и цеолиты в рационах для утят на откорме // Зоотехния. 2002. № 10, C. 18-19. 128 Шадрин А.М., Панин Л.Е. и др. Сахаптин – природный цеолит – уникальная кормовая и профилактическая добавка в корм животным и птице. Изд-во СО РАСХН. НЗПЯОС им. И. В. Мичурина, 2003. 116 с. 129 Шадрин А.М, Синицын В.А., Белоусов Н.М. Роль природных и модифицированных цеолитов в профилактике кормовых и экологических стрессов у животных и птиц // Сибирский вестник сельскохозяйственной науки, 2006. № 6. С. 43-49. 130 Шахмурзов М.М. Охрана рыбохозяйственных водоемов при загрязнении азотсодержащими соединениями и пути снижения их токсичности для рыб: дисс. д-ра биол. наук. М., 1994.

134

131 Шахмурзов М.М., Дацерхоев В.М., Тлупов Т.Х. Оптимизация химического режима воды для получения экологически чистой рыбной продукции // Вестник ветеринарии, 1998. № 8(2). С. 15-18. 132 Кузнецов А.А. Рыбоводно-биологическая эффективность применения природного цеолита-клиноптилолита в составе комбикормов для радужной форели и сибирского осетра: дисс. канд. биол. наук. М., 2002. 22 с. 133 Дацерхоев В.М. Использование природных цеолитов для снижения уровня солей тяжелых металлов в воде и выращивания экологически чистой рыбной продукции: дисс. канд. биол. наук. Ставрополь, 1999. 134 Кулаев С.Н. Эффективность использования природных цеолитов в комбикормах для карпа: дисс. канд. с.-х. наук. Краснодар, 2002. 135 Поляков А.Д., Бузмаков Г.Т., Рассолов С.Н. Транспортировка личинок карпа с использованием цеолита (на примере Кемеровской области) // Успехи соврем. естествознания. 2009а. № 6. C. 66-67. 136 Поляков А.Д., Бузмаков Г.Т., Рассолов С.Н. Использование цеолитового туфа в качестве добавки в рацион сеголетков карпов // Соврем. наукоем. технологии. 2009б. № 2. C. 35-37. 137 Канидьев А.Н., Лабутин В.Г. Эффективность добавления в комбикорм радужной форели природного цеолита (клиноптилолита) // Вопросы интенсификации прудового рыбоводства: Науч. сб. М.: ВНИИПРХ, 1985. - Вып. 45. - С. 178-184. 138 Таратухин В.А., Шимульская Л.К. Корма для карпа с добавкой цеолитового туфа// Рыбн. хоз-во. 1984. - Вып. 9. - С. 35-36. 139 Rimar, J., Gavalova, Z. 2002. Utilization of calcium formate as an ingredient in compound feed, as well as a preservative for silage forage. Chemko Strazske 16, 121. 140 Skalicka, M., Korenekova, B., Makoova, Z., Nad, P. 2002. Effect of additives on the selected poultry production indicators. Zbornýk z mezdinarodnej vedeckej konferencie. pp. 237. 141 FAO, Food and agriculture organization of the United States. Fish is food for the brain as well as good protein. FAO USA, 2000 http://www.fao.org/focus/e/fisheries/nutr.htm 142 Arannilewa S.T., Salawu S.O., Sorungbe A.A., Ola-Salawu B.B. Effect of frozen period on the chemical, microbiological and sensory quality of frozen tilapia fish (Sarotherodun galiaenus) // African Journal of Biotechnology. – 2005. - №4. – P. 852-855. 143 Andrew A.E. Fish processing technology. – Nigeria: University of Llorin press, 2001. – 7-8 pp. 144 FAO, Fisheries and Aquaculture Department, Proteins from fish and fish products. FAO, 2014 http://www.fao.org/fishery/topic/14869/en. 145 FAO, United Nations Food & Agriculture Organization, Nutritional elements of fish. FAO Rome, 2005. 146 Hryniewiecki L. Białka. In Zywienie człowieka. Podstawy nauki o zywieniu. – Warszawa: Wydawnictwo Naukowe Warszawa, 2000. - 176–192 pp.

135

147 Usydus Z., Szlinder-Richert J., Adamczyk M. Protein quality and amino acid profiles of fish products // Food Chemistry. – 2009. - №112. – P. 139-145. 148 Papes F., Surpili J. S., Langone F., Trigo J. R., Arruda P. The essential amino acid lysine acts as precursor of glutamate in the mammalian central nervous system // FEBS Letters, - 2001. - №488. – P. 34–38. 149 Pieniak Z., Verbeke W., Brunsø K., Scholder J. How European consumers perceive fish in terms of health benefits and safety risks // In 37th WEFTA annual meeting Lisbon. - Lisbon, 2007. - P. 3-13. 150 Schaafsma G. Protein digestibility – corrected amino acid score // Journal of Nutrition, - 2000. - №7. – P. 1865–1867. 151 Chalamaiah M., Dinesh Kumar B., Hemalatha R., Jyothirmayi T. Fish protein hydrolysates: Proximate composition, amino acid composition, antioxidant activities and applications: A review // Food Chemistry, - 2012. - №4. – P. 3020- 3038. 152 Chalamaiah M., Balaswamy K., Narsing Rao G., Prabhakara Rao P., Jyothirmayi T. Chemical composition and functional properties of mrigal (Cirrhinus mrigala) egg protein concentrates and their application in pasta // Journal of Food Science and Technology, 2011. - №3. – P. 514-520. 153 Kim J., Lall S. Amino acid composition of whole body tissue of Atlantic halibut (Hippoglossus hipoglossus), yellowtail flounder (Pleuronectes ferruginea) and Japanease flounder (Paralichthys olivaceus) //Aquaculture, 2000. - №187. P.367–373. 154 Schlechtriem C., Fliedner A., Schäfers C. Determination of lipid content in fish samples from bioaccumulation studies: contributions to the revision of guideline OECD 305 // Environmental sciences Europe, 2012. – 155 Guler G.O., Kiztanir B., Aktumsek A., Citil O.B., Ozparlak H. Determination of the seasonal changes on total fatty acid composition and ω3/ω6 ratios of carp (Cyprinus carpio L.) muscle lipids in Beysehir Lake (Turkey) // Food Chemistry, 2008. - №108. P. 689–694. 156 Ćirković M., Đorđević V., Milošević N., Ljubojević D., Babić J. Meat quality of tench in different conditions of production // VII međunarodni gospodarsko znanstveni skup o ribarstvu: Hrvatsko ribarstvo ne pragu EU. Ribakao funkcionalna hrana. – Vukovar, 2011. – 342 p. 157 Ćirković M., Jovanović B., Maletin S. Ribarstvo. - Novy Sad: Universitetu Novom Sadu, 2002.- 231-234. 158 Özyrt G., Polat A., Loker G. B. Vitamin and mineral content of pike perch (Sander lucioperca) common carp (Cyprinus carpio), and European catfish (Silurus glanis) // Turkish Journal of Veterinary and Animal Sciences, 2009. - №4. P. 351– 356. 159 Ackman R. G. Nutritional composition of fats in seafoods // Progress in Food and Nutrition Science, 2000. №13. P.161–241. 160 Buchtova H., Svobodova Z., Križek M., Vacha F., Kocour M., Velišek J. Fatty acid composition in intramuscular lipids of experimental scaly crossbreds in 3- year-old common carp (Cyprinus carpio L.) // Acta Veterinaria Brno, 2007. - №76. P. 73–81.

136

161 Zakes Z., Jankowska B., Jarmołowicz S., Zmijewski T., Partyka K., Demska-Zakes K. Effects of different dietary fatty acids profiles on the growth performance and body composition of juvenile tench (Tinca tinca (L.)) // Reviews in Fish Biology and Fisheries, 2010. - №20. P. 389–401. 162 Caballero M. J., Obach A., Rosenlund G., Montero D., Gisvold M., Izquierdo M. S. Impact of different lipid sources on growth. Lipid digestibility tissue fatty acid composition and histology of rainbow trout. Oncorhynchus mykiss // Aquaculture, 2002. - №214. P. 253–271. 163 Domaizon D. C., Debroas D., Bourdier G. Influence of zooplankton and phytoplankton on the fatty acid composition of digesta and tissue lipids of silver carp: mesocosm experiment // Journal of Fish Biology, 2000. - №57. P. 417–432. 164 Vladau V.V., Bud I., Stefan R. Nutritive value of fish meat comparative to some animals meat // Bulletin UASVM Animal Science and Biotechnologies, 2008. -№65. P. 301–305. 165 Trbović D., Vranić D., Đinović J., Borović B., Spirić D., Babić J., Spirić A. Masnokiselinski sastav i sadržaj holesterola u mišićnom tkivu jednogodišnjeg šarana (Cyprinus carpio) u fazi uzgoja // Tehnologija mesa, 2009. -№50. P. 276–286. 166 Ćirković M., Trbović D., Milošević N., Đorđević V., Janković S., Ljubojević D. Meat quality of two years-old tench and carp grown in extensive conditions // XIV International Symposium Feed Technology. - Novi Sad, 2010. – 257 p. 167 Piironen V., Toivo J., Lampi A.M. New data for cholesterol contents in meat, fish, milk, eggs and their products consumed in Finland // Journal of Food Composition and Analysis, 2002. - №15. P. 705–713. 168 Li G., Sinclair A., Li D. Comparison of lipid content and Fatty Acid composition in the edible meat of wild and cultured freshwater and marine fish and shrimps from china // J Agric Food Chem, 2011. - №5. P. 1871-1881. 169 Weaver K.L., Ivester P., Chilton J.A., Wilson M.D., Pandey P., Chilton F.H. The content of favorable and unfavorable polyunsaturated fatty acids found in commonly eaten fish // J Am Diet Assoc, 2008. - №7. P. 1178-1185. 170 Wall R., Ross R.P., Fitzgerald G.F., Stanton C. Fatty acids from fish: the anti-inflammatory potential of long-chain omega-3 fatty acids // Nutr Rew, 2010. - №5. P. 280-289. 171 Siriwardhana N., Kalupahana N.S., Moustaid-Moussa N. Health benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid and docosahexaenoic acid // Ady Food Nutr Res, 2012. - №65. P. 211-222. 172 Miller M.R., Nichols P.D., Carter C.G. n-3 Oil sources for use in aquaculture--alternatives to the unsustainable harvest of wild fish // Nutr Res Rev, 2008. - №2. P. 85-96. 173 Suriah A.R., Teh S.H., Osman N., Nik M.D. Fatty acid composition of some Malaysian freshwater fish // Food Chemistry, 1995. -№54. P. 45-49. 174 Ugoala C., Ndukwe G.I., Audu T.O. Comparision of fatty acids profile of some freshwater and marine fishes // Internet Journal of food safety, 2008. - №10. P. 9-17.

137

175 Magee A., Julshamn K., Ulgenes Y. A comparision of tissue levels of four essential trage elements in wild and farmed atlantic salmon (Salmo salar) // Fisk Dir Skr, 1991. - №2. P. 111-116. 176 Waagbo R. et.al. Dietary vitamin C, immunology and disease resistance in Atlantic salmon (Salmo salar) // Ponencia presentada en el IV International Symposium on fish nutrition and feeding, Biarritz, Francia, 24-27 de junio de 1991. 177 Fawole O.O., Ogundiran M.A., Ayandiran T.A., Olagunju O.F. Proximate and in some selected fresh water fishes in Nigeria // Internet Journal of Food Safety, 2007. - №9. P. 52-55. 178 Benbrook C.M. Organochlorine Residues Pose Surprisingly High Dietary Risks. J. Epidemiol. Community Health 2002; 56 822. 179 Lehotay S.J., Mastovska K., Yun S.J. Evaluation of Two Fast And Easy Methods For Pesticide Residue Analysis In Fatty Food Matrices. J. Aoac Int. 2005; 88 630. 180 Qiu X., Zhu T., Yao B., Hu, J. Hu, S. Contribution of Dicofol To The Current Ddt Pollution In China. Environ.Sci. Technol. 2005; 39 4385. 181 Bilrha, H., Roy, R., Moreau, B., Belles-Isles, M., Dewailly, E., Ayotte, P.,. In vitro activation of cord blood mononuclear cells and cytokine production in a remote coastal population exposed to organochlorines and methyl mercury. Environ. Health Perspect. 2003; 111 182 Lavoie E. T. and Grasman, K. A. Effects of in ovo exposure to PCBs 126 and 77 on mortality, deformities and post-hatch immune function in chickens, J. Toxicol. Environ. Health, Part A 2007, 70 547. 183 Bernhoft A., Skaare J.U.,Wiig O. Possible immunotoxic effects of organochlorines in polar bears (Ursus maritimus) at Svalbard, J. Toxicol. Environ. Health, Part A, 2000; 59 561. 184 Repetto R., Baliga S.S. Pesticides and immunosuppression: the risks to public health, Health Policy Plan., 1997; 12 97. 185 Ahlborg U.G., Lipworth L., Titus-Ernstoff L. Organochlorine compounds in relation to breast cancer, endometrial cancer, and endometriosis: an assessment of the biological and epidemiological evidence, Crit. Rev. Toxicol., 1995; 25 463. 186 Hardell L., Liljegren G., Lindstrom G. Increased concentrations of chlordane in adipose tissue from non-Hodgkin’s lymphoma patients compared with controls without a malignant disease. Int J Oncol 1996; 9. 187 Morgan D.P., Roan C.C. Absorption, storage, and metabolic conversion of ingested DDT and DDT metabolites in man. Arch Environ Health 1971; 22 301. 188 Cocco P., Brennan P., Ibba A., de Sanjose L.S., Maynadie M., Nieters A., et al. Plasma polychlorobiphenyl and organochlorine pesticide level and risk of major lymphoma subtypes. Occup Environ Med 2008; 65 132. 189 Bocquene G., Franco A. Pesticide contamination of the coastline of martinique. Mar. Pollut. Bull. 2005; 51 612. 190 Gilbert-Lopez B., Garcia-Reyes J.F., Molina-Díaz A. Sample treatment And determination of pesticide residues in fatty vegetable matrices: A review. talanta 2009; 79 109.

138

191 Ledoum M. Analytical methods applied to the Determination of pesticide residues in foods of animal origin. A review of the past two decades. J. Chromatogr. A 2011; 1218 1021 192 Veierov D. Aharonson N. Improved Cleanup Of Large Lipid Samples For Electron Capture Gas Chromatographic Quantitation And Gas Chromatographic- Mass Spectrometric Confirmation Of Organochlorine Residues. J. Assoc. Off. Anal. Chem. 1980; 63 202. 193 Young S.J., Kamps L.R. Gas-Liquid Chromatographic Determination Of Nonpolar Organochlorine Pesticide Residues In A Crude Vegetable Oil And Its Refinery By Products. J. Assoc. Off. Anal. Chem. 1982; 65 916. 194 Luke M.A., Forberg J.E., Masumoto H.T. Extraction And Cleanup Of Organochlorine, Organophosphate, Organonitrogen, And Hydrocarbon Pesticides In Produce For Determination By Gas-Liquid-Chromatography. J. Assoc. Off. Agric. Chem. 1975; 58 1020. 195 Stefanelli P., Santilio A., Cataldi L., Dommarco R. Multiresidue Analysis Of Organochlorine And Pyrethroid Pesticides In Ground Beef Meat By Gas Chromatography-Mass Spectrometry. J. Environ. Sci. Health B 2009; 44 350. 196 Sannino A., Mambriani P., Bandini M., Bolzoni L. Multiresidue Method For Determination Of Organochlorine Insecticides And Polychlorinated Biphenyl Congeners In Fatty Processed Foods. J. Aoac Int. 1996; 79 1434. 197 Lazaro R., Herrera A., Arino A.A., Conchello M.P., Bayarri J. Organochlorine Pesticide Residues In Total Diet Samples From Arago ́N (Northeastern Spain). Agric.Food Chem. 1996; 44 2742. 198 Patel K., Fussell R.J., Hetmanski M., Goodall D.M., Keely B.J. Evaluation Of Gas Chromatography-Tandem Quadrupole Mass Spectrometry For The Determination Of Organochlorine Pesticides In Fats And Oils. J. Chromatogr. A. 2005; 1068 289. 199 Schenck F.J., Donoghue D.J. Determination Of Organochlorine And Organophosphorus Pesticide Residues In Eggs Using A Solid Phase Extraction Cleanup. Agric.Food Chem. 2000; 48 6412. 200 Muralidharan S., Dhananjayan V., Risebrough R., Prakash V., Jayakumar R., Bloom P.H. Persistent Organochlorine Pesticide Residues In Tissues And Eggs Of White-Backed Vulture, Gyps Bengalensis From Different Locations In India. Bull. Environ.Contam. Toxicol. 2008; 81 561. 201 Ghidini S., Zanardi E., Battaglia A., Varisco G., Ferretti E., Campanini G., Chizzolini R. Comparison Of Contaminant And Residue Levels In Organic And Conventional Milk And Meat Products From Northern Italy. Food Addit. Contam. 2005; 22 9. 202 Bennett D.A., Chung A.C., Lee S.M. Multiresidue Method For Analysis Of Pesticides In Liquid Whole Milk. J. Aoac Int. 1997; 80 1065. 203 Hong J., Kima H.Y., Kim D.G., Seo J., Kimb K.J. Rapid Determination Of Chlorinated Pesticides In Fish By Freezing-Lipid Filtration, Solid-Phase Extraction And Gas Chromatography-Mass Spectrometry. J. Chromatogr. A 2004; 1038 27. 204 Garridofrenich A., Martinezvidal J., Cruzsicilia A., Gonzalezrodriguez M., Plazabolanos P. Multiresidue Analysis Of Organochlorine And Organophosphorus

139

Pesticides In Muscle Of Chicken, Pork And Lamb By Gas Chromatography–Triple Quadrupole Mass Spectrometry. Anal. Chim. Acta 2006; 558 42. 205 Cunha S.C., Lehotay S.J., Mastovska K., Fernandes J.O., Beatriz M., Oliveira P.P. Evaluation Of The Quechers Sample Preparation Approach For The Analysis Of Pesticide Residues In Olives. J. Sep. Sci. 2007; 30 620. 206 Tao S., Liu W.X., Li X.Q., Zhou D.X., Li X., Yang Y.F., Yue D.P., Coveney R.M. Organochlorine Pesticide Residuals In Chickens And Eggs At A Poultry Farm In Beijing, China; Environ. Pollut. 2009; 157 497. 207 Covaci A., Gheorghe A., Schepens P. Distribution of organochlorine pesticides and alpha-Hch enantiomers in pork tissues.Chemosphere 2004; 56 757. 208 Doong R.A., Lee C.Y. Determination Of Organochlorine Pesticide Residues In Foods Using Solid-Phase Extraction Clean-Up Cartridges. Analyst 1999; 124 1287. 209 Muralidharan S., Dhananjayan V., Jayanthi. Organochlorine Pesticides In Commercial Marine Fishes Of Coimbatore, India And Their Suitability For Human Consumption. P. Environ. Res. 2009; 109 15. 210 Campos A., Lino C.M., Cardoso S.M., Silveira M.I. Organochlorine Pesticide Residues In European Sardine, Horse Mackerel And Atlantic Mackerel From Portugal. Food Addit. Contam. 2005; 22 642. 211 Fidalgo-Used N., Centineo G., Blanco-Gonzalez E., Sanz-Medel A. Solid- Phase Microextraction As A Clean-Up And Preconcentration Procedure For Organochlorine Pesticides Determination In Fish Tissue By Gas Chromatography With Electron Capture Detection. J. Chromatogr. A 2003; 1017 35. 212 Hopper M.L. Automated One-Step Supercritical Fluid Extraction And Clean-Up System For The Analysis Of Pesticide Residues In Fatty Matrices. J. Chromatogr. A 1999; 840 93.

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Application A Pesticide residue chromatograms

Fig.1. EC chromatograms of cereal samples after MSPD extraction and LLE extraction: 14 pesticides on one detector – 1. HCB (5.964); 2. alpha-HCH (6.991); 3. gamma-HCH (7.960); 4. heptachlor (8.522); 5. aldrine (9.273); 6. beta-HCH (9.813); 7. heptachlor-epoxide (11.305); 8. p,p’ DDE (12.892); 9. dieldryna (13.652); 10. endrin (14.660); 11. o,p’ DDT (15.048); 12. p,p’ DDD (16.388); 13. p,p’ DDT (16.381); 14. DMDT (18.198)

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Fig.2. EC chromatograms of cereal samples after MSPD extraction and LLE extraction: 18 pesticides on one detector – 1. cymoxanil; 2. pencycuron; 3.pencycuron 4. chlorpyrifos methyl; 5. chlorpyrifos ethyl; 6. formothion; 7. cyanazine; 8. myclobutanyl; 9. propargit; 10. oksadixyl; 11. phosalone; 12. fenarimol; 13. cyfluthrin; 14. cyfluthrin; 15. cyfluthrin; 16. tau-fluvalinate; 17. azoxystrobin; 18.promaquizafop

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Fig.3. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 17 pesticides on one detector – 1. cymoxanil; 2. pencycuron; 3. DEET; 4. pyrimethanil; 5. chlorpyrifos methyl; 6. chlorpyrifos ethyl; 7. formothion; 8. cyprodinil; 9. isofenphos-ethyl; 10. cyanazine; 11. myclobutanyl; 12. oksadixyl; 13. lenacil; 14. phosalone; 15. fenarimol; 16.azoxystrobin; 17.promaquizafop

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Fig.4. EC chromatogram of cereal samples after MSPD extraction and LLE extraction: 18 pesticides on one detector – 1. propham; 2. diazinon; 3.zooxamide; 4. parathion-ethyl; 5. mecarbam; 6. methidathion; 7. flutriafol; 8. bupirimate; 9. quinoxygen; 10. metamitron; 11. iprodione; 12. iprodione; 13. acrinathrin; 14. acrinathrin; 15. prochloraz; 16. acetamiprid; 17. dimethomorph; 18. Dimethomorph

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Fig.5. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 19 pesticides on one detector – 1. propham; 2. heptenophos; 3. diazinon; 4. simazine; 5.pirimicarb; 6. chlorpyrifos methyl; 7. metalaxyl; 8. chlorpyrifos ethyl; 9. parathion-ethyl; 10. mecarbam; 11. methidathion; 12. flutriafol; 13. bupirimate; 14. quinoxyfen; 15. fenarimol; 16.triazophos; 17. metamitron; 18. iprodion; 19. prochloraz; 20. acetamiprid

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Fig.6. EC chromatogram of cereal samples after MSPD extraction and LLE extraction: 21 pesticides on one detector – 1. dichlorphos; 2. teflubenzuron; 3.propachlor; 4. quintozene; 5. acetochlor; 6. paraoxon- methyl; 7. malathion; 8. pendimethalin; 9. tetrachlorvinfos; 10. buprofezin; 11. oxyfluorfen; 12. ethion; 13. fipronil; 14. DFF; 15. bromopropylate; 16. dimoxystrobin; 17. metconazole; 18. azinophos-methyl; 19. azinophos-ethyl; 20. alpha-cypermethrin; 21. alpha-cypermethrin

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Fig.7. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 13 pesticides on one detector – 1. propachlor; 2. atrazine; 3.acetochlor; 4. prometrine; 5.paraoxon-methyl; 6. malathion; 7. pendimethalin; 8. tetrachlorvinfos; 9.buprofezin; 10. ethion; 11. dimoxystrobin; 12. metconazole; 13. azinphos-methyl; 14. azinphos-ethyl

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Fig.8. EC chromatogram of cereal samples after MSPD extraction and LLE extraction: 18 pesticides on one detector – 1. tecnazene; 2. propyzamide; 3.metribuzin; 4. bromophos-methyl; 5. acetochlor; 6. alfa endosulfan; 7. folpet; 8.captan; 9. fluorochloridon; 10. nitrofen; 11. beta-endosulfan; 12. endosulfan sulfate; 13. tetradifon; 14. beta-cyfluthrin; 15. beta-cyfluthrin; 16. beta-cyfluthrin; 17. esfenvalerate; 18. esfenvalerate

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Fig.9. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 12 pesticides on one detector – 1. propyzamide; 2. cyprazine; 3. metribuzin; 4. bromophos-methyl; 5. carbaryl; 6. metazachlor; 7. mepanipyrim; 8.tiabendazol; 9. iprovalicarb; 10. iprovalicarb; 11. benalaxyl; 12. pyriproxyfen

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Fig.10. EC chromatogram of cereal samples after MSPD extraction and LLE extraction: 17 pesticides on one detector – 1. cadusafos; 2. fenchlorphos; 3.paraoxon-ethyl; 4. chlorfenvinphos; 5. picoxystrobin; 6. kresoxim-methyl; 7. bifenthrin; 8. fenhexamid; 9. fluazynam; 10. lambda cyhalothrin; 11. cypermethrin; 12. cypermethrin; 13. cypermethrin; 14. fenvalerate; 15. fenvalerate; 16. indoxacarb

150

Fig.11. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 11 pesticides on one detector – 1.cadusafos; 2. propoxur; 3. clomazone; 4. fenchlorphos; 5. fenitrothion; 6. paraoxon- ethyl; 7. chlorfenvinphos; 8.picoxystrobin; 9. kresoxim-methyl; 10. cyprconazole; 11. tebufenpyrad

151

Fig.12. EC chromatogram of cereal samples after MSPD extraction and LLE extraction: 17 pesticides on one detector – 1. vinclozolin; 2. dichlofluanid; 3.triadimefon; 4. tolylfluanid; 5. procymidone; 6. imazalil; 7. azaconazole; 8. trifloxystrobin; 9. fenpropathrin; 10. fenamidon; 11. permethrin; 12. permethrin; 13. zeta cypermethrina; 14. zeta cypermethrina; 15 zeta cypermethrina; 16. deltamethrin; 17. deltamethrin

152

Fig.13. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 15 pesticides on one detector – 1. diphenylamine; 2.chloropropham; 3.carbofuran; 4. fenpropimorph; 5. vinclozolin; 6. dichlofluanid; 7. triadimefon; 8. tolylfluanid; 9. procymidone; 10. napropamide; 11. imazalil; 12. azaconazole; 13. trifloxystrobin; 14. fenpropathrin; 15 fenamidon

153

Fig.14. EC chromatogram of cereal samples after MSPD extraction and LLE extraction: 21 pesticides on one detector – 1. mevinphos; 2. trifluralin; 3.propazine; 4. tolclofos-methyl; 5. malaoxon; 6 dicofol; 7. bromophos-ethyl; 8. penconazole; 9. tetraconazole; 10. triadimenol; 11. . triadimenol; 12. paclobutrazol; 13. diniconazole; 14. propiconazole; 15. bromuconazole; 16. bromuconazole; 17. fluquinconazole; 18.bitertanol; 19. fenbuconazole; 20. dimifenoconazole; 21. imibenconazole

154

Fig.15. NP chromatogram of cereal samples after MSPD extraction and LLE extraction: 21 pesticides on one detector – 1. mevinphos; 2.trifluralin; 3.propazine; 4. tolclofos-methyl; 5. malaoxon; 6. bromophos- ethyl; 7. penconazole; 8. tetraconazole; 9. triadimenol; 10. triadimenol; 11. paclobutrazol; 12. flusilazole; 13. diniconazole; 14. propiconazole; 15. propiconazole; 16. fludioxonil; 17. tebuconazole; 18. bromuconazole; 19. fluquinconazole; 20. bitertanol; 21.fenbuconazole; 22. difenoconazole

155

Application B

156

Application C

157