Comprehensive Diagnosis of Selenium and Iodine Deficiencies in Golden Manchurian Quails and Gray-Speckled Guinea Fowls Risen in the Astrakhan Region

| This study presents the results of comprehensive diagnosis of combined selenium-iodine hypomicroelementosis in quails and guinea fowls adapting to new biogeochemical conditions. This study focused on golden Manchurian quails, Coturnix japonica (Temminck and Schlegel, 1849), and gray-speckled guinea fowls, Numida meleagris (Linnaeus, 1758), imported to the Astrakhan region in 2015 from the Krasnodar Krai, Russia. The major components of terrestrial ecosystem (soil, water, and plants) in the Astrakhan region were found to be low in selenium, iodine and cobalt. As a result, quails and guinea fowls received a diet with insufficient amounts of therein. All subjects were examined for microelements, physiological, hematological, and biochemical changes, and pituitary-thyroid hormones. In addition, the study examined antioxidant indicators and markers of free radical oxidation. The comprehensive examination revealed selenium deficiency and iodine deficiency in the body of examined quails and guinea fowls. The experimental birds were found to have elevated levels of blood corpuscles, glucose, diene conjugates, and malonic aldehyde. Levels of antioxidant vitamins (E, A, C) were found to be low. Based on the above, it can be concluded that golden Manchurian quails and gray-speckled guinea fowls raised in conditions of selenium deficiency and iodine deficiency can experience latent symptoms of hypomicroelementosis when fed a diet based on local plants. The consequence is a drop in egg productivity. Thus, farmers specialized at breeding quails and guinea fowls should considers feeds enriched with selenium and iodine when the local ecosystem lacks a sufficient amount of therein.

for quails and guinea fowls is bird feed. This may pose a challenge since birds with high metabolic rates have increased sensitivity to important trace element deficiencies in plant-based feeds. The consequences include a decline in immune function, oxidative stress, and latent forms of hypomicroelementosis, conditions caused due to the insufficient intake of essential trace elements. Normally, these health issues do not have pronounced clinical manifestations, and indicators such as body temperature, pulse, and respiratory rate are within the normal range. However, there are evidences that birds suffering from trace element deficiency experience decrease in productivity, encounter reproductive problems (Corrie, 1996;Fisinin, 2012;Tsurik and Bezborodov, 2015;Temiraev et al., 2016, Vorobiev et al., 2018, and have difficulty adapting to new conditions (Fisinin, 2012;Tsurik and Bezborodov, 2015;Vorobiev et al., 2018). Thus, it is important to diagnose the latent form of hypomicroelementosis in birds in time. However, the fact that this condition proceeds without clinical symptoms significantly complicates the diagnosis and necessitates the conduct of clinical biochemical study, including at the molecular-cellular level (Rodionova et al., 2010;Kostin, 2017).
The negative effects of stress can cause pathological changes in the body of birds. It was established that reactions such as free radical oxidation and antioxidant defense are indicative for that kind of process (Belyaev, 2011). The free radical oxidation involves radical cations of selenium, iodine, manganese, zinc, copper, cobalt, molybdenum, and iron-sulfur clusters (Rodionova et al., 2010, Vorobiev, 2013. When there is a shortage of selenium in the body, the activity of antioxidant enzymes decreases. This interferes with the neutralization of hydroperoxides and lipid peroxides and results in the development of oxidative stress (Weiss et al., 1990). Selenium affects the thyroid function and its deficiency can aggravate the functional and structural changes in the thyroid gland. Since selenium works in conjunction with iodine, the low selenium level can cause the iodine absorption to worsen, resulting in iodine deficiency (Weiss et al., 1990;Braun et al., 1991). Selenium supports the conversion of thyroid hormone thyroxine (T4) into its active analogue triiodothyronine (T3). The latter participates in the synthesis of thyroglobulin, which accelerates the conversion of carotene into vitamin A (Struev and Simakhov, 2006). Other consequences of selenium deficiency are reproductive failure, increased propensity to inflammatory diseases, and decrease in immune function. The iodine deficiency, on the other hand, affects the thyroid hormone production, resulting in metabolic abnormalities.
Overall, selenium deficiency and iodine deficiency are conducive to asymptomatic hypomicroelementosis and slower metabolism. This is the reason why it is impossible to reach the full genetic potential of poultry birds (Vorobiev, 2013;Vorobiev et al., 2018).
The molecular and cellular mechanisms underlying the pathogenesis of asymptomatic hypomicroelementosis in birds are poorly studied. There is insufficient evidence about the diagnosis of latent hypomicroelementosis in poultry birds, especially quails and guinea fowls. Studies only provide sketchy information about selenium and iodine deficiencies in chickens and some other domesticated birds (Kostin, 2017;Gorelik and Derkho, 2013;Kolesnik and Derkho, 2015;Popova et al., 2015).
In recent years, Russian farmers have been actively importing quails and guinea fowls into the Lower Volga region, where terrestrial ecosystems were found to have low levels of selenium and iodine (Samokhin, 2008;Vorobiev, 2013). The insufficient intake of selenium and iodine can negatively affect the adaptive capacity and productivity of birds. However, there are no comprehensive studies on hypomicroelementosis induced by deficiency of combined selenium and iodine in imported quails and guinea fowls. As a result, there are no evidence about many important health indicators for birds that were imported to the Astrakhan region, such as microelement profile, metabolism, hematological parameters, free radical oxidation, antioxidant activity, pituitary-thyroid function, trace element content of feed, and egg productivity. This study aims to close this gap. Following the above mentioned, the main hypothesis of this study implies that low level of selenium and iodine in the main components of terrestrial ecosystems of the Lower Volga region (soils, water, plants) and local plant forages predetermines the development of selenium-iodine deficiency in acclimatized agricultural birds, which negatively affects metabolism and many physiological and biochemical parameters of birds, reducing their productivity.
This study aimed to perform a comprehensive diagnostics of combined selenium-iodine hypomicroelementosis in quails and guinea fowls acclimatized in biogeochemical conditions of terrestrial ecosystems in the Astrakhan region.

MAtErIAlS And MEthodS
This study focused on golden Manchurian quails, Coturnix japonica (Temminck and Schlegel, 1849), and gray-speckled guinea fowls, Numida meleagris (Linnaeus, 1758), imported to the Astrakhan region in 2015 from the Krasnodar Krai, Russia. Between 2015 and 2017, samples from multiple sources were collected using sampling methods according to Kovalski and Maclyanaya (1982). For comparison, samples were taken from farms both in the Astra-

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June 2020 | Volume 9 | Issue 6 | Page 906 khan and Krasnodar regions. Overall, the study included 36 soil samples, 15 water samples, 31 samples collected from plants and plant-based feed to measure the trace element content, and 186 tissue samples from internal organs of quails and guinea fowls farmed in the Astrakhan region (experimental birds). The comparison tissue samples were taken from 10 quails and 10 guinea fowls raised in the Krasnodar Krai (control birds). Data from Vorobiev et. al. (2018) was used to assess the selenium and iodine content in the ecosystem components of the control region (Krasnodar Krai). All animal experiments and diagnostic tests were carried out in accordance with the European Community Council Directives (86/609/ EEC) and the Declaration of Helsinki.
A comprehensive diagnostic study of combined selenium and iodine deficiency was carried out in 2016 at the farm in the Kamyzyak district, Astrakhan region. The study used 20 four-month-old quails (10 females and 10 males, weighing 298 ± 7.1 g and 261 ± 8.3 g, respectively) and 20 nine-month-old guinea fowls (10 females and 10 males, weighing 2.9 ± 0.31 kg and 2.8 ± 0.56 kg, respectively). Five females and males of four-month-old quails (weighing 296± 6.4 g and 264 ± 7.2 g, respectively) and five females and males of nine-month-old guinea fowls (weighing 3.1 ± 0.42 kg and 2.7 ± 0.48 kg, respectively) from a farm in Labinsky District, Krasnodar Krai, from where agricultural birds were brought to Astrakhan Region, were studied as control birds.
Quails were kept in battery cages with 5 birds in each cage, whereas guinea fowls were housed in the floor space of a typical house with 10 birds in each. Experimental birds were fed based on recommendations of the All-Russian Research and Technological Institute of Poultry. The following measurements were taken from the birds through clinical examination: body temperature, pulse rate, respiratory rate, and egg productivity. The content of metal elements such as Cu, Mn, Zn, and Co in tissue samples was measured by electrochemically by atomic absorption (Britske, 1982) using a SHITACHI 180-50 spectrophotometer. The selenium content was determined fluorometrically according to Nazarenko and Ermakov (1971). Iodine determination was performed by the rhodanate-nitrite method.
The hematological analysis provides information about the following measurements: red blood cell (RBC) count, white blood cell (WBC) count, leukocyte formula, erythrocyte sedimentation rate (ESR), hemoglobin, glucose, alkali reserve, total protein, uric acid, and total lipids. All experiments were performed using generally accepted methods (Kondrakhin et al., 2004). For this, blood samples were collected by puncture of the brachial vein before feeding. The total calcium and inorganic phosphate in blood plasma were determined according to the method of Kalnitsky (1980). The vitamin E content in the blood serum samples was estimated by Emmerie and Engel reaction with ferrous dipyridyl complex using the Minichrom chromatograph equipped with scanning UV detector. The vitamin A content was measured by the reaction with antimony chloride (Dvinskaya et al., 1979). The vitamin C content was determined as described by Petrova et al. (1987).
Diene conjugate (DC) levels in the blood serum of birds were detected by UV spectroscopy (Platzer et al., 1970). Malondialdehyde (MDA) levels were estimated by the method of Buzlama et al. (1997) The catalase (CAT) activity was measured as described by Korolyuk (1998). The superoxide dismutase (SOD) enzyme activity was measured determined by measuring its ability to compete with nitro blue tetrazolium for superoxide anions according to the method of Chevari et al. (1985). The glutathione peroxidase (GPx) activity was determined by the procedure of Paglia and Valentine (1967). The function of the endocrine system was evaluated through measuring thyroid-stimulating hormone (TSH), total thyroxine (T4) and total triiodothyronine (T3) on the Uniplan analyzer using the enzyme linked immunosorbent assay as described by Matreshina et al. (1998). For this, ELISA-AT-T and Adrenocorticotropic Hormone (ACTH) ELISA kits (Biomerica, Inc.) were employed. The results were read on a vertical scanning spectrophotometer at 450 nm.
Statistical data processing of was carried out using Microsoft Excel Pro and Statistica. The Student's t-test was used to measure the reliability of difference between the two means. Differences were considered significant at P <0.05.

rESultS And dIScuSSIon microelement contentS of internal organS aS a diagnoStic tool
It is important to measure the amount of trace elements in soil, water and plants in the study site because these concentrations define the trace element content of bird feed. Comparing biogeochemical characteristics of the Astrakhan region and sites with chernozem soils (Vorobiev, 1993;Matveev et al., 1997;Ermakov et al., 2008;Kostin, 2017), it was found that the major components of terrestrial ecosystem (soil, water, and plants) in the Astrakhan region are low in selenium, iodine and cobalt. No deficiency of manganese, zinc, iron, and copper was detected. Hence, the bird feed made from local plants will have low contents of selenium (between 0.04 ± 0.006 and 0.12 ± 0.08 mg/kg), cobalt (between 0.01 and 8.9 ± 0.37 mg/kg) and iodine (between 0.01 and 0.07 ± 0.004 mg/kg).
Since plants absorb essential elements directly from soil, the level of selenium, iodine, copper, manganese, zinc and

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June 2020 | Volume 9 | Issue 6 | Page 908 cobalt in plants is dependent upon their content in the soil (r = + 0.59 to r = + 0.65). The type and physiology of the plant are also important. One of the crucial markers for hypomicroelementosis in quails and guinea fowls are microelements, which occur in insufficient concentrations in the terrestrial ecosystem of the breeding farm.
Estimating the content of trace elements in tissue samples from experimental birds (Tables 1-2), it was found that studied elements were present in the body of quails and guinea fowls in the following decreasing order: Zn > Mn > Cu > Co > Se ≥ I. Similar results were obtained for other poultry birds (Vorobiev et al., 2018). The selenium and iodine levels were found to vary greatly amongst samples (Table 1).
In female quails, for example, selenium was found in the following organs and tissues, as arranged in order of decreasing concentration: liver > whole egg > spleen > intestinal wall > egg yolk > blood > egg shells > egg white > feathers > blood ≥ ovaries ≥ muscles ≥ heart muscle. Organs and tissues of male quails can be arranged in the order of decreasing selenium content as follows: feathers ≥ semen ≥ testicles > heart muscle > intestinal wall > spleen > muscles > blood. The iodine content in the examined female quails decreased in the following order: egg yolk ≥ liver > blood > egg white > ovaries > spleen ≥ intestinal wall > heart muscles > muscles > egg shells. For male quails, this order is as follows: testicles > sperm > liver > intestinal wall > blood ≥ spleen > heart muscle > feather > muscles.
The results of the microelement analysis for female and male guinea fowls are similar (Table 2).
Organs and tissues of female guinea fowls can be arranged in the order of decreasing selenium content as follows: egg yolk > liver > intestinal wall > blood > cardiac muscle > spleen ≥ egg shell > muscles > feathers. For iodine, the decreasing order is liver ≥ ovaries > egg yolk ≥ egg white ≥ shell ≥ intestinal wall > blood > muscles > feathers.
The overall accumulation of microelements in guinea fowls and quails farmed in the Astrakhan region was somewhat similar given the deficiency of I, Se, and Co in the terrestrial ecosystem. However, there is a difference in microelement assimilation. Quails accumulate significantly more deficient microelements (Se, J and Co) than guinea fowls (P <0.05). This can be explained by the fact that quails that have higher metabolic rate (Fisinin, 2012). Other factors include feed storage, and feed composition, and fodder cultivation method.
The comparison of microelement contents between experimental and control birds showed that birds in the Astrakhan region received significantly less diet selenium and iodine (P < 0.05). This finding correlates with the low amount of selenium and iodine in the terrestrial ecosystem of the region (Tables 1-2). The low selenium and iodine levels are one of the biomarkers for hypomicroelementosis. It is likely that selenium deficiency negatively affects the synthesis of antioxidant enzymes, especially glutathione peroxidase, the molecule of which contains the given trace element. The iodine deficiency negatively affects the synthesis of thyroxine (T4), which increases cardiac output and nerve excitability, and regulates the plumage growth and normal development of feathers (Kostin, 2017).
What is interesting is that the cobalt content of organs and tissues in experimental birds is comparable to that in controls (Tables 1-2). At the same time, the amount of cobalt in terrestrial ecosystem of the Astrakhan region is significantly lower when compared to Krasnodar Krai. Therefore, there is no positive correlation between cobalt content in the environment and cobalt content in the body of the examined birds. This coincides with the previous study (Kostin, 2017), where concentrations of trace elements were examined on pigeons living in conditions of cobalt deficiency.
Based on the above, it can be stated that quails and guinea fowls raised in biogeochemical conditions similar to those recorded in the Astrakhan region will have enough essential microelements, such as zinc, manganese, copper and cobalt. At the same time, they will suffer from iodine and selenium deficiencies. The expected consequences are oxidative stress and asymptomatic hypomicroelementosis.

PhySiological and biochemical ParameterS aS diagnoStic toolS
The analysis of blood revealed that RBC count and WBC count in adult quails and guinea fowls was higher than normal (Table 3) (Motuzko et al., 2008).
The RBC count for adult quails was found to be in the range of 5.97 ± 0.08 million/µL x 10 12 /L. The RBC count for adult guinea fowls was in the range of 7.39 ± 0.09 million/µL x 10 12 /L. The WBC count for adult quails was found to be in the range of 14.02 ± 1.27 thousand/µL x 10 12 /L. The WBC count for guinea fowls was in the range of 13.9 ± 1.4 thousand/µL x 10 12 /L. The blood glucose levels varied in quails and guinea fowls within the range of 15.06 ± 1.04 mmol/L and 13.1 ± 1.7 mmol/L (Table 4), respectively.
Based on the results of biochemical analysis, it can be concluded that biochemical profile is suitable for determining hypomicroelementosis in golden Manchurian quails and gray-spotted guinea fowls caused by the combined effect of selenium and iodine deficiencies.

free radical oxidation and antioxidant activity in the blood aS diagnoStic toolS
The indicators of lipid peroxidation (LPO) and antioxidant defense against continues stress, incl. microelement deficiency, serve as biomarkers of adaptation and home-ostasis. The antioxidant defense system consists of exogenous (Ca, vitamins E, A, and C, Se, etc.) and endogenous (CAT, SOD, and selenium-containing GPx) antioxidants (Lankin et al., 2001). Exposure to diet-induced stress is associated with a sharp decline of vitamins, such as A, D and B2, in poultry. In stressful conditions, domesticated birds need for 4 times more vitamins E and C and potassium (Papazyan et al., 2009;Fisinin, 2012). The vitamin E plays a protective role in lipid peroxidation and is closely linked to selenium (Lankin, 2001;Rodionova et al., 2010;Vorobiev, 2013).
The amount of free radical oxidation products, such as diene conjugates (DC) and malondialdehyde (MDA), and carbohydrates in the blood of experimental quails and guinea fowls was significantly higher than in controls (P <0.05). This is in line with previous studies. The CAT, SOD and GPx activities in experimental poultry were sig

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June 2020 | Volume 9 | Issue 6 | Page 910  nificantly lower than in control quails and guinea fowls (P <0.05). This coincides with the existing findings (Skorkina et al., 2004;Popova et al., 2015;Temiraev et al., 2016). An increase in the levels of DC and MDA can be considered as a destructive event, which causes damage to cell membranes. Finally, quails had significantly higher blood levels of total lipids, DC, MDA, CAT, SOD, and vitamins E and A (Table 5) when compared to guinea fowls (P <0.05).
Based on the above results, it can be concluded that the examined species of poultry have a latent form of hypomicroelementosis, which was caused by the combined effect of selenium and iodine deficiencies.

Pituitary-thyroid hormone levelS aS diagnoStic toolS
To confirm the diagnosis of latent combined selenium-iodine hypomicroelementosis in the examined birds, TSH, T4, and total T3 levels were measured. It is known that thyroid hormones are responsible for post-stress recovery, enhance oxygen consumption by the tissues of the body, and are closely linked to iodine and selenium. Although iodine deficiency in the body was previously found to inhibit egg productivity in different species of poultry (Fisinin, 2012; Gorelik and Derkho, 2013;Ermakov et al., 2018), there are no comprehensive studies on the diagnosis of hypomicroelementosis in quails and guinea fowls using pituitary-thyroid function as a reference. Therefore, it is impossible to fully compare data obtained in this study with those in the existing literature. However, the results are comparable with those concerning the pituitary-thyroid function of other species of poultry. For instance, it was found that a situation where there are a relatively high TSH level and relatively low T4 and T3 levels is typical for various species of poultry (Samokhin, 2008;Kolesnik and Derkho, 2015;Temiraev et al., 2016), including quails and guinea fowls (Table 6).
It should be noted that overall, quails had higher hormone level than guinea fowls (P <0.05). Perhaps, this is due to the fact that quails have better metabolism (Fisinin, 2012). It is believed that egg productivity is influenced by the thyroid function. In other words, egg productivity is associated with the basal metabolic rate, regulated by T4, T3, iodine, and selenium (Fisinin, 2012;Gorelik and Derkho, 2013;Kolesnik and Derkho, 2015;Vorobiev et al., 2018). Thus, given the role of selenium and iodine in the functioning of the pituitary-thyroid system, it is logical to assume that the shortage of these microelements in the terrestrial ecosystem and diet can result in a decrease of egg productivity. For quails, the value of egg productivity was in the range of 23.2 ± 0.9 eggs per month, guinea fowls 27.5 ± 1.1 eggs per month. These figures only relate to experimental birds.

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June 2020 | Volume 9 | Issue 6 | Page 911 concluSIonS The comprehensive biogeochemical analysis of terrestrial ecosystems in the Astrakhan region showed low concentrations of selenium, iodine and cobalt in soil, water, and vegetation. As a result, the local diet of quails and guinea fowls contains insufficient amounts of these essential trace elements. Thus, the etiology and pathogenesis of combined selenium and iodine deficiency in the examined birds were caused by the shortage of selenium and iodine in the major components of terrestrial ecosystem and diet. The comprehensive examination revealed physiological and biochemical changes in the body of quails and guinea fowls imported to the Astrakhan region. There were the elevated levels of blood corpuscles, glucose, diene conjugates, and malonic aldehyde. Levels of antioxidant vitamins (E, A, C) in experimental birds were found to be low. The CAT, SOD, and GPx activates decreased in response to combined selenium and iodine deficiency. At the same time, levels of hemoglobin, total protein, total lipids, P, Ca, Zn and Mn were at the lowest limit of normal. Based on the above, it can be concluded that golden Manchurian quails and gray-speckled guinea fowls raised in conditions of selenium deficiency and iodine deficiency can experience latent symptoms of hypomicroelementosis when fed a diet based on local plants. The consequence is a drop-in egg productivity. Physiological and biochemical parameters of blood, indicators of metabolism status, levels of POL and AOS, and the activity of pituitary-thyroid system (TTG, T4, T3) provide an opportunity for correction and prevention of this asymptomatic disease of the studied birds, which are in biogeochemical conditions of Se and J deficit in the main components of ecosystems. It allows for a scientifically grounded diagnosis and choice of the missing trace elements in plant foods and bodies of quails and guinea fowls in order to apply them for improvement of metabolism and increase of integrative functions of egg production in birds.
Thus, farmers specialized at breeding quails and guinea fowls should considers feeds enriched with selenium and iodine when the local ecosystem lacks a sufficient amount of therein. It will improve the metabolism of quails and guinea fowls, increase egg production and weight of eggs of farm birds, which is a cost-effective measure. According to the previous study, good results were obtained by enriching feed with organic preparations DAFS-25 in a dose of 1.6 mg/kg and YODDAR in the amount of 50 g/t. (Polkovnichenko et al., 2019).