The Strategic Application of Electrolyte Balance to Minimize Heat Stress in Broilers

Several physiological and metabolic changes are triggered in broilers submitted to high environmental temperatures, resulting in performance losses. Feed formulation manipulation of the dietary electrolyte balance may be applied to reduce the negative impact of heat stress on broiler performance. This experiment was carried out to evaluate the effect of the manipulations of dietary electrolytes by combining changes in the electrolyte (Na++K+–Cl-) balance (EB) and in the [(K++Cl-)/Na+] ratio (ER) in broiler feeds. In total, 1575 male broilers between 21 and 46 days old were allotted to 15 treatments in a 5x3 factorial arrangement, consisting of five diets with different EB/ER combinations (150/3, 250/2, 250/3, 250/4, and 350/3). Birds were submitted to heat stress at 25 or 35 days old. Live performance, mortality rate, and carcass traits were evaluated. The strategic formulation of diets with different EB and ER improves live performance and minimize the effect of heat stress on broilers. Under thermoneutral conditions, an EB of 250 mEq/kg and an ER of 3 are recommended, whereas under heat stress, and EB of 350 mEq/kg and an ER of 3 should be applied.


IntRoduCtIon
Broiler susceptibility to heat stress increase as air relative humidity and environmental temperature values exceed the thermal comfort zone (16-23ºC and 50-70%) (Tinoco, 1998), making it difficult for birds to dissipate heat.Consequently, their body temperature increase, negatively affecting their performance.
The addition of salts to the drinking water or to the feed is frequently employed in broiler production to reduce losses caused by heat stress.The main salts used are potassium chloride (KCl) and sodium bicarbonate (NaHCO 3 ) (Cahaner & Leenstra, 1992;Yalçin et al., 2001).
The minerals K + , Na + and Cl -, in particular, play essential roles in metabolism due to their participation in the osmotic balance, in the acid-base balance, and in the integrity of mechanisms that regulate the transport of substances across the cell membranes.Imbalances among those minerals have a direct effect on acid-base balance, affecting many metabolic functions, and therefore, broiler performance (Judice et al., 2002).
The environment and the diet influence the acid-base balance.Maintaining this balance is essential to improve the performance of broilers reared under high temperatures and to overcome the damaging effects of respiratory alkalosis produced by heat stress (Teeter & Belay, 1996).
Previously, only the acid-base balance (K + , Na + and Cl -) minimum requirements for each rearing phase was previously considered in diet formulation (NRC, 1994), However, according to Mongin (1981), the proportions (difference and ratio) among those minerals should be adjusted to provide better electrolyte balance with the aim of maintaining physiological acid-base homeostasis, and thereby, optimal growth performance (Gezen et al., 2005).Mongin (1981) also emphasizes that diets should contain not only an adequate electrolyte balance, given by the difference (Na + +K + -Cl -), but also adequate electrolyte ratio [(K + +Cl -)/Na + ].Therefore, the adequate ratio between K + , Na + and Cl -needs to be considered in addition to the calculation of the difference between the total concentration of anions and cations (Talbolt, 1978).
The present experiment was carried out to experiment was carried out to evaluate the effect of the manipulations of dietary electrolyte balance based on combinations of different electrolyte balances and electrolyte ratios on the performance, mortality, and carcass traits of broilers submitted to heat stress.

MAteRIAl And MethodS
The experiment was carried out at the Animal Science Experimental Sector of the School of Veterinary Medicine of Universidade Estadual Paulista (UNESP), Araçatuba campus, state of São Paulo, Brazil.In total, 1575 male broilers belonging to the same commercial genetic strain, were evaluated between 21 and 46 days of age.
Birds were housed in a masonry broiler house (7.85 x 45.70m) built in the East-West direction, equipped with an adiabatic evaporative cooling system and negative-pressure ventilation, and covered with special tiles made of insulating material (expanded polystyrene) placed under reflecting metal plates.Birds were distributed in floor pens (1.4x3.0m) with wood-shavings litter, and equipped with automatic feeders and drinkers.Each pen was considered as an experimental unit.
At 21 days of age, chicks were weighed and randomly distributed to the pens, which housed 35 birds each.A lighting program of 18 hours of light (12 hours of natural light + six hours of artificial light) was applied.Birds were fed with five diets consistingwith ofdifferent electrolyte balances, inaccording to accordance with the subsequent treatments, and keptmaintained under in thermoneutral conditions.
Treatments consisted of diets containing five different electrolyte balances, derived from the EB/ ER combinations of 150/3, 250/2, 250/3, 250/4, and 350/4, shown in Table 1, and thermal environments (thermoneutral conditions or acute heat stress).Acute heat stress was or not applied when birds were 25 or 35 days old, and fed the same diet of other treatments.Evaluations were carried out when birds were 33 (day 33) and 46 (day 46) days old.On day 33, data were analyzed according to completely randomized experimental design in a 5x2 factorial arrangement (five EB/ER combinations x two environmental temperatures), totaling 10 treatments (Table 2), with six replicates of birds maintained under thermoneutral conditions and three replicates of birds submitted to heat stress on day 25.Each replicate contained 35 birds.On day 46, responses were analyzed according to completely randomized experimental design in a 5x3 factorial arrangement was applied, totaling 15 treatments (Table 2), with three replicates of 35 birds each.The model of the analysis of variance is presented in Table 3.The experimental diets were based on corn, soybean meal, corn gluten, soybean oil, vitamin and mineral supplement, calcitic limestone, and dicalcium phosphate, and formulated according to the recommendations of Rostagno et al.( 2005) (Table 4).Salt (NaCl, NaHCO 3, K 2 SO 4 ) levels were included to supply minimum K + , Na + and Cl -requirements and adjusted according to the electrolyte combinations of each experimental diet.Feed was manufactured in the experimental feed mill of the Animal Science Experimental Sector, UNESP.

The Strategic Application of Electrolyte Balance to Minimize Heat Stress in Broilers
Birds were submitted, according to treatment (33 or 46 days of age), to acute heat stress (Gonzalez-Esquerra & Leeson, 2005), mimicking commercial production conditions when there is power outage, and consequently cooling and evaporation systems stop working.Under these conditions, birds suffer from heat stress due to high environmental temperature and air relative humidity, in addition to high ammonia concentrations.Temperature was maintained between 35-38°C for 4h on days 25 and 35by covering the pens corresponding to the heat-stress treatment with a transparent plastic canvas and providing a heat source (porcelain cones with a 600w resistance) controlled by a thermostat.Birds were offered feed and cold water ad libitum during the entire experimental period, including the days when heat-stress was applied.
The following performance parameters were evaluated on days 33 and 46: body weight gain (g/bird/ period), feed intake (g/bird/period), feed conversion ratio, and mortality rate.Feed conversion ratio was calculating by dividing feed intake by weight gain.All dead birds were weighed and their body weight used to correct feed conversion ratio.During the heat-stress periods, mortality was recorded according to treatment, body weight, and age.Dead birds were disposed in a compost pile located in the experimental facilities.
The following micro-climatic conditions inside the broiler house and at the pens: air temperature (dry-bulb temperature), black globe temperature, air relative humidity, and air velocity.Average microclimatic parameters inside the house were obtained by an automated weather station placed at the center of the house.Air temperature and relative humidity were detected by a sensor placed inside a weatherresistant shelter with reading ranges of -40 to 60ºC for temperature and 0 to 100% for relative humidity.Air velocity was determined with the aid of an ultrasonic anemometer, with 0-60 m.s -1 sensitivity and 0.01 m.s - 1 resolution.All records were managed and stored using a multi-channel data acquisition system (CR10, Campbell Scientific).Temperature and relative humidity were monitored using the Ibutton® in five pens (except for air velocity, which was measured in a single pen) among the birds in treatments not submitted to heat stress.In birds submitted to heat stress, those parameters were measured only on the heat stress days in five pens (one per electrolyte balance treatment).
Five birds per experimental unit (15 birds per treatment), with body weight close to the average of the respective replicate, were selected on day 46 for carcass yield evaluation.These birds were sacrificed, plucked, and eviscerated.The abdominal fat pad (adipose tissue around the bursa, proventriculus, gizzard, and cloaca) was removed and its weight calculated relative to broiler weight at sacrifice.The eviscerated carcass, with no head, feed, and neck, was weighed again to calculate carcass yield relative to live weight.The carcass was then cut up, and the yield of the bone-in parts (whole breast, legs (thighs and drumsticks), and wings) was calculated relative to carcass yield.
The obtained parts and the remaining carcass were ground, and sample was collected and freeze-dried for total lipid composition analysis.The lipid content of the carcass samples was determined by extraction in Soxhlet apparatus, using ethyl ether as solvent.
Data were submitted to analysis of variance to verify the effect of treatments, and then to analysis of regression to evaluate the effects of each supplementation level, according to the PROC GLM of SAS system (SAS Institute, 2000).Means were compared by the Student's t-test.
All parameters described in percentages were normalized for statistical analysis by transformation in arcsine 100 X .

ReSultS And dISCuSSIon
Maximum average environmental temperatures were 37.3 and 29.5ºC on the first and second heatstress days, respectively.The remaining of the house was under thermoneutral conditions (19ºC -62%).
Live weight, feed intake, and feed conversion ratio were influenced by heat stress.Birds submitted to heat stress both on day 25 and day 45 presented reduced live weight, increased feed intake, and worse feed conversion ratio (Table 5).These results are consistent with the observations of Plavnik & Yahav (1998), who reported progressive reduction of body weight, weight gain, feed intake, and feed efficiency of broilers submitted to high environmental temperatures.This worse performance may be explained by the fact that, in an attempt to adapt to heat stress, birds reduce their feed intake to try to reduce endogenous heat production (Teeter et al., 1985).Bonnet et al.(1997) concluded that broilers submitted to heat stress gained 50% less weight than those maintained in thermoneutral conditions.

The Strategic Application of Electrolyte Balance to Minimize Heat Stress in Broilers
higher than 1 in broiler diets, but does not provide any precise figure.Therefore, further studies on this subject are needed (Borgatti et al., 2004).
In the present experiment, the diets with an EB of 250 mEq/kg resulted in better feed conversion ratio (Table 5).However, the results show that diet formulation should also take into account ER, because the broilers fed the experimental diet with 250 mEq/kg and an ER value of 3 presented better feed conversion ratio relative to those fed the other diets, both on days 33 and 46.
Because linear feed formulation does not allow the use of ratios, because division is unfeasible in the software, it was not possible to apply of the full concept of Mongin (1981), that is, to supply in the diet, simultaneously:1-minimum Na + , K + e Cl -requirements, 2-EB of 250 mEq/kg, and 3-ER >1.This has probably limited the research on dietary electrolyte balance for many years.However, the recent publication of the PPFR spreadsheet (Garcia Neto, 2011), which applies a non-linear concept, allows formulation changes such as those applied in the present experiment, and therefore, to check for nutritional imbalances, as reported by Ravindran et al. (2008).Unbalanced ratios among K + , Cl -, and particularly Na + , may limit the absorption of amino acids and other nutrients (Ravindran et al.,.2008).This problem may be circumvented by the application of the electrolyte combination (both EB and ER) concept, thereby preventing imbalances among electrolytes and their consequences on broiler growth.
The results of the present experiment show that broilers fed diets with adequate electrolyte balance were more tolerant to heat stress.The broilers submitted to acute heat stress at 25 and 35 days of age (±36°C for 4 h) and fed diets with an EB/ER of 350/3:1 dealt more efficiently with this extreme impact, as shown by their lower mortality rate (Table 6).
When broilers are submitted to acute heat stress, in addition to high temperature and high humidity, they are also exposed to high ammonia levels.The association of these three factors may have been the cause of the high mortality observed in this experiment, as it is difficult to separate their individual effects on bird survival.However, the mortality rate of the broilers fed a diet with the combination of an electrolyte balance of 350 mEq/kg and an electrolyte ratio of 3:1 was clearly lower, demonstrating that these birds were capable of maintaining their better acid-base homeostasis and suffered less changes caused by acute stress, including respiratory alkalosis.
The importance of supplying optimal Na+, K+ e Cl-levels in broiler diets is that, during heat stress, the competition between H+ e K+ in the renal tubules is reduced.During alkalosis, extracellular K+ enters the cell, consequently increasing the secretion of this ion in the renal tubule lumen.Under these conditions, H+ is exchanged by K+ in the renal tubule.The increasing K+ secretion reduces its blood concentration, which causes circulation disorders in broilers, leading to death.
Table 5 -Effects of different combination of electrolyte balance (Na + +K + -Cl -) and electrolyte ratio [(K + +Cl -)/Na + ] in the diet of broilers submitted to acute heat stress with 25 or 35 days old on their weight gain (WG), feed intake (FI), and feed conversion ratio (FCR) between 1 and 46 days of age.

The Strategic Application of Electrolyte Balance to Minimize Heat Stress in Broilers
Table 6 -Effects of the combination of electrolyte balance (Na++K+-Cl-) with electrolyte ratio [(K++Cl-)/Na+] in the diet of broilers submitted to acute heat stress with 25 or 35 days old on the daily intake (g) of potassium (K), sodium (Na), and chorine (Cl), and on the mortality rate of 1-to 46-d-old male broilers.Temim et al., 2000b;Faria Filho, 2003) These findings are associated with the different metabolism of each specific tissue: the breast present glycolytic metabolism, whereas the leg muscles present oxidative metabolism (Ain Baziz et al., 1996).
The broilers exposed to heat stress on day 36 presented higher carcass fat content between 22 and 46 days of age compared with the broilers submitted to heat stress on day 25 and with those not exposed to heat stress.The higher fat content and lower crude protein content of the broilers submitted to heat stress (Cheng et al., 1997a,b) results from their lower basal metabolism and physical activity (Ain Baziz et al., 1996).
The findings of the present experiment indicate that ER may potentially improve broiler performance and carcass quality, provided EB is concurrently adjusted, particularly under heat stress conditions (Table 7).The birds fed the diet with an EB of 250 mEq/kg and an ER of 3 were heavier at slaughter and presented heavier carcasses and lower abdominal fat content compared with those submitted to the other treatments.This is an important factor to be considered because consumers value heavy and lean chicken carcasses.
Considering that broilers exposed to high temperatures tend to habituate to these conditions, and that those temperatures negatively affect their productivity (Yahav & McMurtry, 2001), it is essential to supply them a nutritionally adequate diet that provides support to prevent disorders caused by heat stress.For instance, there may be reduced weight gain, which may be subsequently compensated (Yahav & McMurtry, 2001), and directed for body fat deposition and visceral growth or for protein accretion (Zhan et al., 2007;Rutz, 2011).

ConCluSIonS
The strategic formulation considering the precise electrolyte balance, including EB and ER, improves broiler performance and may prevent the negative effects of heat stress.
Under thermoneutral conditions, an EB of 250 mEq/ kg and an ER of 3 are recommended, whereas under heat stress, an EB of 350 mEq/kg and an ER of 3 to obtain better performance and livability.

Table 1 -
Electrolyte balance (EB) and electrolyte ratio (ER) combinations applied in the experimental diets.

Table 2 -
Treatments according to electrolyte balance and ratio combinations and thermal environment.

Table 3 -
Model of the analysis of variance of the experiment.

Table 4 -
Ingredients and calculated composition of the diets fed to the broilers during the grower and finisher phases.

Garcia Neto M, Perri SHV, Faria Júnior MJ de A, Pinto MF
Means followed by the same letter in the same column, within each factor, are not different by the Student's t-test at 5% probability level.

Gamba JP, Rodrigues MM, Garcia Neto M, Perri SHV, Faria Júnior MJ de A, Pinto MF
a-e Means followed by the same letter in the same column, within each factor, are not different by the Student's t-test at 5% probability level.*= interaction between dietary electrolyte combination (EB/ER) and acute heat stress.