Scientific Article Dietary electrolyte balance on serum hematological and biochemical parameters of broilers under heat stress

The aim of this study was to evaluate the effect of different dietary electrolyte balance (DEB) levels over the hematological parameters and the biochemical profile of broilers under stress caused by cyclic heat. We used 1050 male broilers from Ross lineage, for two experimental phases: phase 1 (broilers from 1 until 21 days old) and phase 2 (broilers from 22 until 42 days old). The broilers were distributed under a completely randomized design, built for five treatments (110; 175; 240; 305; 370 mEq of DEB/kg of ration) and seven repetitions. For the hematological analyses were used two broilers in each repetition, one to blood count evaluation (Hemogram) and another one to the biochemistry evaluation, collected in the end of each experimental phase. During the phase of 1 - 21 days old, the variable hemoglobin number (Heme), mean corpuscular volume (MCV) and Serum Chloride (Cl) presented a linear effect (p<0,05) DEB levels. There was no effects (p>0,05) over the corpuscular volume (CV), total plasma protein (TPP), leukocytes number (Leu), heterophiles (Het), lymphocytes (Lyn), eosinophils (Eos), basophils (Bas), monocytes (Mon), calcium (Ca), phosphor (P), uric acid (UA) e urea (U). During the phase two of 22 – 42 days old there was effect (p<0,05) quadratic to the variables Leu and Het and linear to the variable P and significative difference (p<0,05) between the variable means UA at the levels of DEB tested. There was no effect (p>0,05) over the variable CV, Heme, MCV, TPP, Lyn, Eos, Bas, Mon, Cl, Ca and U on the DEB levels tested. The supplementation of the DEB levels 110, 175 e 240, 305 and 370 to the phase from 1 until 21 days old presented great results in the hematological evaluation and biochemical, because it provided possibly a longer useful life to the Hem without promoting a metabolic imbalance. In the phase from 22 until 42 days old the level of 268 to 280 mEq of DEB/kg of ration presented a better answer from the broilers immune systemic.


Introduction
One of the biggest obstacles about broilers creation is the environment in which they grow up, mainly in tropical and subtropical regions where predominate high temperatures, in which the chronic heat stress (over 30 ºC) can harm poultry production [1]. As soon as the ambient temperature exceeds the thermal comfort temperature of the broiler, where the metabolic rate is minimal and the homeotherm is maintained with less energy expenditure, the heat stress begins [2]. Consequently, the broilers can suffer metabolic imbalance (acidosis or alkalosis), decreased feed consumption and low growth rate, impaired health and increased mortality [3].
Respiratory alkalosis occurs in the heat stress situation due to an increase in the respiratory rate, resulting in excessive carbon dioxide losses (CO 2 ). Thus, the partial pressure of CO 2 (pCO 2 ) decreases, leading to a drop in the concentration of carbonic acid (H 2 CO 3 ) and hydrogen (H + ). On the other hand, as the stress intensifies, there is an increase in the partial pressure of (pCO 2 ) with an increase in the level of H + , causing respiratory acidosis [4].
In tropical production systems, several strategies are used to mitigate the effects of heat stress, such as modifying the environment through the projection of acclimatized facilities, managements such as feeding and temperature control of drinking water, in addition to changes in the diet composition in order to promote greater intake or compensate for low food consumption with or without the use of additives such as vitamins and minerals [5].
Dietary electrolyte supplementation has been used to maintain the acid-base balance in the blood, minimizing the deleterious effects of heat stress [6]. Mongin [7] and Johnson and Karunajeewa [8] were the first ones to relate the applicability of the electrolytes use, with the intent of balance their lose from the broilers under heat stress, these authors observed better performance in broilers fed with diets under dietary electrolyte balance (DEB) 250 and 180 to 300 mEq / kg, respectively. Ahmad et al [6] found out that a DEB between 50 and 250 mEq / kg promotes acidobasic normalization of broilers under heat stress, however, DEB of 0 mEq / kg causes growth depression due to lower feed consumption, because of blood acidosis, and the DEB 350 mEq / kg, causes alkalosis, associated with a high level of NaHCO 3 [6].
In the hematological and biochemical evaluation, it has already been observed that heat stress causes an increase in hematocrit (justified by the effect of dehydration caused by vomiting and diarrhea) and in the number of red blood cells through an increase in erythropoiesis [9]. The H/L ratio rises as a consequence of the heterophile's increase and reduction of lymphocyte, this relationship is a sensitive index of chronic stress in broilers [11]. There has also been observed a decrease in serum total plasma proteins and uric acid, an increase and/or decrease in chlorine, among other changes [12,13].
Therefore, the evaluation of the efficiency of the inclusion of DEB can be carried out through blood parameters, since these parameters are very susceptible to temperature variation, constituting into an important indication of physiological responses to stress [11].
Based on these evidences, the effect of the electrolyte balance (110; 175; 240; 305; 370 mEq/kg) on the hemogram and the serum biochemical profile of broilers will be evaluated, in order to identify the efficiency of these diets in situations of cyclic heat stress.

Materials and methods
This study was conducted in strict accordance with the recommendations of the guide for care and use of laboratory animals from the National Institutes of Health. The protocol was approved by the Ethics Committee on Animal Experimentation at the Federal University of Piauí (Piauí, Brazil) under the number 075/15.

Birds and diets
The experiment was conducted in the poultry sector of the Federal University of Piauí, located in Bom Jesus-PI, Brazil (latitude: 9° 4′30″ south and longitude: 44° 21′26″ west). 1050 male Ross broilers were used for two experimental phases: phase 1 (broilers from 1 to 21 days old) and phase 2 (broilers from 22 to 42 days old), 525 in each phase. The animals were distributed in a completely randomized design, consisting of five treatments (levels of electrolyte balance) and seven replications of 15 broilers, and for hematological analysis two broilers from each repetition were used, one for hemogram evaluation and another for biochemical evaluation collected at the end of each experimental phase, 72 per experimental phase, totaling 144 birds.
The experimental diets were formulated to attend the nutritional requirements of the broilers in each phase (1 to 21 and 22 to 42 days), considering the requirements and chemical composition of the ingredients, as described by Rostagno et al. [14], except for sodium, chlorine and potassium that were adjusted to define the electrolyte balances tested, and also to assess the levels of these elements in the ingredients used (Table 1, 2, 3 and 4). To obtain the DEB levels (110; 175; 240; 305; 370 mEq / kg), ammonia chloride (NH 4 Cl), potassium carbonate (K 2 CO 3 ) and/or sodium bicarbonate (NaHCO 3 ) were added to the diets, replacing the inert material. Water and feed were provided ad libitum to the animals.
Throughout the experimental period, a continuous light program (24 hours of natural + artificial light) was adopted, using 60 Watt incandescent lamps, applying thermal stress to these animals, considering that the temperature in Bom Jesus tends to go beyond the thermal comfort zone for broilers, which according to Abreu and Abreu [15] is around 26 to 29 ° C for broilers with 21 days and 20°C for broilers with 42 days, with relative humidity between 60 to 70% for both phases. The temperature and relative humidity of the air inside the shed was measured daily, throughout the experimental period, by thermohygrometers.

Sample collection
For laboratory analysis of the blood count and serum biochemistry, 0.5 to 3 mL of venous blood were collected, depending on the size of the bird and not exceeding 1% of the animal's weight, by puncture of the jugular vein, medial metatarsal or ulnar vein, in syringes containing 10% EDTA anticoagulant (ethylenediaminetetraacetic acid) for blood count and syringes without anticoagulant for biochemical analysis [16]. Blood samples were taken in the two experimental phases, the first phase was performed on the 20th day of age and the second phase on the 41st day of age.

Hemogram
Immediately after collection, blood smears were made, subsequently stained with hematological dye type Romanowsky (Panotic Rapid®) and displacement under an optical microscope, without a 1000x increase, for differential assessment of leukocytes. Then the absolute red blood cell and leukocyte counts were performed in a Neubauer camera using 0.01% toluidine blue. Hematocrit was also determined by centrifuging a blood sample in a microhematocrit capillary and calculating the mean corpuscular volume (MCV) according to Hendrix [17]. Total plasma protein (TPP)

Biochemistry
The determination of TPP was performed after centrifuging blood in a microhematocrit capillary and reading the plasma protein concentration by refractometer [16].

Statistical analysis
The data were subjected to a normality test (Cramer-Von Mises), with analysis of variance (ANOVA) by the GLM procedure of the Statistical Analysis System -SAS software, version 9.0. The SNK test with α = 0.05 probability was used to compare means. Estimates of dietary electrolyte balance were defined using polynomial regression.

Biochemistry
The results of the biochemical parameters are presented in Table 9 and 10 for the phase of 1 -21 and 22 -42 days of age, respectively. For the 1-21 days old phase, the variable chlorine (Cl) showed a linear effect (p <0.05) for the levels of electrolyte balance tested. There was no effect (p> 0.05) on the variables calcium (Ca), phosphorus (P), uric acid (UA) and urea (U) depending on the tested DEB levels. It was observed a value below the species reference for the variable P on all levels tested.  [22], 'Domingues et al. [23], "Ahmad et al. [6], @Thrall et al. [16]. For the phase of 22-42 days of age, the variable P showed a linear effect (p <0.05) in order of the DEB levels tested, there was also a significant difference (p <0.05) between the means UA variable only between the levels of 175 and 305 mEq of DEB/Kg of feed. There was no effect (p <0.05) on the variables Cl, Ca and U due to the tested DEB levels. A value below the species reference was observed for the variable Cl at the level 305 mEq/Kg.

Discussion
Electrolyte supplementation is used to mitigate the harmful effects of heat stress, in order to balance electrolyte losses by birds in thermal stress [6,7,8]. Quantitative and morphological changes in blood cells are associated with infectious, inflammatory conditions and stresses of a diverse nature, as we can see by variations in hematocrit values, number of circulating leukocytes and erythrocytes and hemoglobin content in red blood cells [25].
In a situation of chronic thermal stress, it is expected that there will be a decrease in the total number of red blood cells associated with a shorter lifespan and inhibition of production by the bone marrow, a decrease in MCV and a decrease in the number of leukocytes associated with lower feed consumption and an increase in costicosterone that depresses the activity of lymphoid organs, and a lower level of TPP [26,13].
In previous studies it has been observed that the use of DEB for broilers improves the performance of birds, body weight gain, bone mineralization, decreased mortality and the normalization of the acid-basic balance, making softer the effect of thermal stress keeping hematological and biochemical parameters within the normal range. [10,9,6].
In this study, it was observed that in the phase of 1 -21 days of age there was a linear effect (p <0.05) for the parameters, Heme and MCV, where an increase was observed proportionally to the DEB levels of Heme and an inversely proportional decrease of the MCV, both within the reference value for species, suggesting for this stage that the red blood cells have a longer useful life proportional to the increase in the level of DEB, considering that the red blood cells were increasing in number and decreasing in size. This can be considered, since according to Christian [27], how higher the animal's metabolic rate, bigger the accumulated oxidative stress and the lower the average life of the erythrocytes. What reinforces this observation is that in addition to be increased in number, it was decreasing in size, indicating that it was living longer, even in the senescence process, as observed by Bartosz [28] the older the red blood cell, the smaller its size.
There was also a linear effect (p <0.05) for the level of Cl, in which its level decreases as the DEB increased. It has already been observed that thermal stress decreases the serum level of Cl- [13,10], electrolyte is necessary in body fluids to exert an acidic action to normalize blood pH [11]. Ahmad et al. [6], also observed a linear decrease (p <0.05) in Cl as the DEB increased. In this study, Cl was within the reference value for the species in all DEB levels tested, however the linear decrease in its level indicates that values above the level of 370 would cause a state of hypochloremia, and it is not recommended to use values above this.
Monocytosis and hypophosphatemia were common to all DEB levels tested for this same phase. Monocytosis in birds is common in cases of infectious diseases caused by microorganisms that promote granulomatous inflammation such as Mycobacterium, Chlamydia and fungi, chronic bacterial granuloma and extensive tissue necrosis and zinc deficiency [16], while hypophosphatemia is common in the growth phase of young birds, hypovitaminosis D3 (with hypocalcaemia), malabsorption, starvation and prolonged corticosteroid therapies [16].
No evidence has been identified to justify these two situations then further studies need to be carried out.  [16] it is a rare phenomenon in birds and a similar situation has not been identified in other studies for DEB levels close to this.
On the 22 -42 days old phase, the level of 268 to 280 mEq of DEB/kg of feed, showed a better response from the broilers immune system, because, within this range, the Leu and Het numbers were lower than in the others DEB levels tested and within the normal range for the specie. These values were obtained using quadratic regression equations.

Conclusion
Supplementation with 370 mEq DEB/Kg of feed improves the hematological and biochemical response, it provided an increase in the number of red blood cells, without promoting metabolic imbalance in the phase from 1 until 21 days old. On the phase from 22 until 42 days old, the levels of 268 to 280 mEq of DEB/kg of additional feed improve the response from the broilers immune system.