Effect of the complex of microelements in the form of L-asparaginates on zootechnical parameters and morphometry of bone and muscle tissue of broiler chickens

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Introduction
In poultry farming, minerals are used as one of the ways to increase metabolism in birds [1]. Minerals form part of the molecules of complex organic structures that take part in metabolic processes, including digestion, thus improving absorption and utilization of nutrients in feed [2]. Lack of microelements in feed causes disease of limb joints of farm birds, reducing its reproduction and productivity, worsening the quality of the eggshell [3]. Zinc, copper, manganese, iron, iodine, selenium, and cobalt are important micronutrients in feeding broiler chickens [4]. It is known that vitamin-mineral premixes containing inorganic salts are used in broiler chicken diets [5][6][7][8][9][10][11]. These premixes are beneficial for metabolic processes but have low bioavailability (2 -10% of those taken with feed) and can destroy vitamins. Their utilization is associated with large energy expenditures. Organic compounds of mineral substances in comparison with inorganic ones have higher bioavailability, provide better utilization of metals and positively affect the productive and reproductive functions of poultry [12][13][14][15][16]. One of these compounds is organic mineral complex additive OMEC, which is a chelate complex compound of L-aspartic amino acid with microelements [4,17,18].
This work aimed to study the effect of organic forms of microelements on zootechnical parameters and morphological structure of pectoral muscles and bone tissue of broiler chickens.

Materials and methods
The experiments were carried out using the broiler chickens of the Selection and Genetic Center "Smena 8" cross in the vivarium of Federal Scientific Center "All-Russian Research and Technological Institute of Poultry" of RAS (VNITIP RAS). Five groups (one control and four experimental) of chickens were formed by the method of analogues. Chickens were kept in an experimental cage battery R-15, 35 heads in each group. Broilers of the control group received nutritionally balanced complete feed of cornwheat type, with the addition of inorganic forms of microelements in accordance with the accepted guaranteed norms of their addition to the mineral premixes for poultry. The level of metabolic energy in the feed was 307 kcal/100g (121.86 MJ/kg), the crude protein content was 22.7% from day 1 to day 21. The level of metabolic energy was 323 kcal/100g (13.53 MJ/kg), the crude protein content was 20% from day 22 to day 35. The content of fish meal in the feed was 6.0% from day 1 to day 21, and 4.5% from day 22 to day 35, the level of available phosphorus was 0.43-0.42%. Chickens of the group No.2 received similar feed to the control in terms of nutritional value without the addition of micronutrients premix. Broilers of the group No.3 received mixed fodder with the addition of organic mineral premix OMEC-broiler containing 10% of the accepted norms of microelements in the form of L-asparaginates. Chickens of the group No.4 received mixed fodder with the addition of mineral premix OMEC-broiler containing 12.5% of the accepted norms of microelements in the form of L-asparaginates. Broiler chickens of the fifth group received mixed fodder with the addition of mineral premix OMEC-broiler containing 20% of the accepted norms of microelements in the form of L-asparaginates, Table 1. Conditions of poultry housing and feeding met the recommendations of VNITIP RAS. Broiler chickens were fed with loose mixed fodder, which was produced using the equipment of VNITIP RAS, vitamin premix of industrial production Vitasol was the same for all groups. Veterinary measures were carried out according to the vaccination plan adopted by the farm. During the experiment, the following characteristics were assessed: -live body weight of chickens at the age of 1, 14, 21 and 35 days by individual weighing of all the flock, -livestock livability, -average daily live body weight gain, -consumption and expenses of feed per 1 kg of live body weight gain, -calcium, phosphorus, manganese, iron, copper, and zinc content in bone tissue.
At the end of the experiment, 15 males were selected from each experimental group of broiler chickens for the study, their slaughter was carried out and samples of bone tissue of tibia and pectoral muscles were taken. The content of microelements, potassium and magnesium were determined by atomic absorption spectrometry. The content of sodium was determined by flame photometry. The content of phosphorus was determined by photometry and the content of calcium by complexometry. Histological examination of the bone and muscular tissue was carried out after micropreparations had been made by a special histological technique. This technique included fixing the selected material in 10% buffered neutral formalin, followed by bone decalcification in an electrolyte decalcifying solution produced by Biovitrum and dehydrating using a closed-type vacuum infiltrator Tissue-Tek VIP 5 Junior (an automatic autonomous tissue processor). Dewaxed sections were stained with hematoxylin and eosin. Photographs of the micropreparations were made using the MC-3 (USB-2.0) No. XC1272 digital camera, LCMOS03100KRA camera, and the MCview computer system.

Results and discussion
The results obtained showed that the livability of broiler chickens during the rearing period in all experimental groups, except the group No.2, was 100%, Figure 1. In the experimental group No.2 the livability of chickens is less by 2.86% compared to other groups. The live body weight of broiler chickens in the group No.2 who received mixed fodder without additional microelements was 1.17-14.69% less than that in the control group at the studied age (14, 21 and 35 days). Moreover, with increasing age of chickens, this difference became larger and by the age of 35 days it acquired a statistically reliable character ( Table  2). When organic complex of microelements in the amount of 10%, 12.5% and 20% of the accepted norms was added to feed, the live body weight of chickens at the age of 14 days in the experimental groups Nos.3-5 exceeded that in the control group by 6.24%, 6.63%, 4.80%. At the age of 21 days the excess weight 8.13%, 10.62%, 8.35%, and at the age of 35 days it was 10.81%, 11.35%, 9.52%, respectively.    When analyzing the indicators of the mineral composition of the tibia of broiler chickens, it can be seen that the levels of calcium and phosphorus in the group No.2 become less by 0.35% and 0.91%, respectively, in comparison with group No.1. However, in the groups No.3-5 the levels of calcium and phosphorus increase. As for potassium and sodium, there are no significant differences compared with the indicators of the control group. In the groups No.3-5 the content of potassium has a tendency to increase, and the content of sodium, on the contrary, has a tendency to decrease. In the group No.2 magnesium content becomes significantly less (by 32%) compared with the control. In the groups Nos.3-4 magnesium content increases, and in the group No.5 its content is equal to that of the control, Table 3. The amount of microelements in the group No.2 showed a sharp decrease. Starting from the group No.3 there is a tendency to increase in the amount of iron up to 6.16%; manganese up to 7.51%, copper up to 11.65%, and zinc up to 4.93%, which is evidence of their good absorption and active participation in metabolic processes.

Morphometric parameters of bone tissue
The morphometric study of the histostructures of bone tissue of broilers of experimental groups that consumed feed with different content of organic and inorganic minerals, showed differences between groups. The bone tissue of chickens in group No.1, shows that the periosteal plate has a typical fibrous structure and is tightly attached to the bone. Figure  4 clearly shows the process of osteohistogenesis, which includes an active formation of bone tissue, the intact zone, the zone of columnar cartilage and cartilage destruction are well differentiated. Images of bone tissue of broilers in group No.2 (received feed without micronutrients premix) show the same processes of indirect osteohistogenesis with the formation of bone tissue, Figure 5. The zone of columnar cartilage and cartilage destruction are well differentiated but the activity of this process is reduced, which is manifested by a smaller amount of formed bone tissue.  The histostructures of the bone tissue of chickens of the experimental group No. 5 (receiving 20% organic mineral supplement OMEC as part of the diet), show that the processes of osteohistogenesis are quite active, Figure 8. Trabeculae are formed by densely clustered plates of osteons, the distribution of osteons in the structure of bone beams is regular, forming a sufficiently strong skeleton. These changes correspond to the age of chicks.

Micromorphology of muscle tissue
The histological study of the muscle tissue of broiler chickens of the experimental groups did not show any special differences, Figures 9-11. The muscle tissue of chickens of all experimental groups was formed by uniformly distributed and densely clustered transversely contoured muscle fibers with rare intermuscular clusters of mature adipocytes and frequent vascular lumen of the microcirculatory channel in the endomysium.  Nuclei distributed along the entire length of the muscle fiber are clearly visible on the micropreparation. Perimysium has a typical morphology. There was no detachment of muscle fibers. In the intermuscular space there is a moderate amount of mature adipose tissue with a small number of small-caliber blood vessels.

Conclusions
The results obtained show that with the addition to the diet of broiler chickens of group No.1 minerals in the form of premix, zootechnical parameters correspond to the norm, with good formation of bone tissue. In the group No.2, without additional premixes in diet, zootechnical parameters decreased in comparison with the control group, feed consumption increased and the processes of osteohistogenesis slowed down. With the addition of the organic mineral additive in the amount of 10%, 12.5% and 20% of the accepted norms of OMEC to the diets of broiler chickens, the zootechnical parameters showed good livability of chickens, increased live body weight, decreased feed expenses per one head, increased average daily gain of chickens with low conversion of feed and EPI. Analysis of indices of the mineral composition of the tibia of broiler chickens of these groups showed that macronutrients in the bone tissue of the tibia of broiler chickens in the group No.2 compared with the control group became less, but starting from the group No.3 their amount increases. The use of complex of microelements in organic form with their higher bioavailability helps to reduce the input levels of iron, manganese, copper, zinc to 10%, 12.5% and 20% of the guaranteed norms. The morphometric data obtained showed weakening of osteohistogenesis processes in the group No.2 of broiler chickens not receiving an additive feed in the form of premixes and the enhancement of these processes in groups Nos. 3-5 receiving the additive complex of organic microelements in the form of L-asparaginate. The data obtained show that the best result is observed in the groups Nos. 3 and 4 receiving 10% and 12.5% of the organic mineral supplement OMEC in the form of Lasparaginate. At the same time, zootechnical parameters of growing broilers and bone mineralization remain at a high level.