The effects of protein , amino acid , and dietary electrolyte balance on broiler chicken performance and blood parameters under heat stress

The effect of crude protein (CP), amino acid (AA), and dietary electrolyte balance (DEB) were evaluated on blood parameters, carcass traits, and broiler performance under heat stress (29-34°C). A total of 540 male chickens (Ross 308) were allocated to 12 diets with factorial arrangement 2 × 2 × 3, using a completely randomized design with three replicates of 15 chickens in grower (13 to 26 days) and finisher (27 to 42 days) periods. and 120, 220, and 320 mEq kg DEB. The level of 21% CP increased body weight gain (BWG) and decreased feed conversion ratio (FCR) at grower period (p < 0.05). In contrast, 20% CP level decreased BWG and increased FCR at finisher period (p < 0.05). Further, 20% CP level reduced blood sodium and blood electrolyte balance (p < 0.05). The highest blood electrolyte balance was achieved by DEB 320 mEq kg diet (p < 0.05). Broiler response to DEB in heat stress depended on the age of bird, length of exposure to high temperature and CP level of the diet. Under heat stress (29-34°C), the 21% CP level at grower period and 17% CP level at finisher period improved broiler BWG and FCR.


Introduction
Nutrition and metabolism play an important role in the acid-base balance of broiler chickens (Borges, Silva, Maiorka, Hooge, & Cummings, 2004;Olanrewaju, Wongpichet, Thaxton, Dozier, & Branton, 2006) and eventually performance.Heat stress is one of the most problems in tropical regions to broiler production.Monovalent electrolytes, i.e., sodium (Na + ), potassium (K + ), and chloride (Cl − ) are the key minerals, which consider as dietary electrolyte balance (DEB, Na + + K + -Cl -) (Mongin, 1981;Borges, Silva, Ariki, Hooge, & Cummings, 2003a), have the greatest impact on acid-base balance or pH of blood and tissues under heat stress (Borges et al., 2004).High temperature accomplish with negative balance of minerals led to broiler respiratory alkalosis, decreased feed intake and growth performance (Belay & Teeter, 1993).The birds under heat stress fed diets with DEB of around 250 mEq kg -1 showed better performance, well maintained blood physiological parameters [pH, HCO 3 , pCO 2 , hemoglobin (Hb), hematocrit (H), lymphocytes (L), and H to L ratio] and blood nutrients (glucose, Na, K and Cl) and also retained more strong ions (Na, K and Cl) (Ahmad & Sarwar, 2006).On the other hand, the growth responses of broilers that consuming diets containing low CP at optimal requirement levels of limited AA (Lys, Met, and Thr) had similar to broiler performance fed diets based on National Research Council (NRC, 1994) recommendations (Safamehr, Narimani, & Nobakht, 2012).Alternatively, DEB can affect nutrient metabolism, especially AA, in broiler (Murakami, Rondon, Martins, Pereira, & Scapinello, 2001).Reduction of nitrogen in poultry wastes and reduce environment pollution is primary consideration of these diets.So, CP levels in diets is depends on limited AA which supplemented in the diets.Therefore, it is necessary to be existent a balance among DEB, AA, and CP levels under heat stress to insure that optimal growth and well-being of broiler were attained.Keeping in view the role of strong ions to alleviate heat stress in broiler chicken and their different AA and CP requirements in this situation, a few publications investigated their interaction on broiler performance.Thus, an experiment was carried out to study the effects of crude protein, supplemental amino acids, and dietary electrolyte balance levels on some blood parameters, carcass traits and broiler performance under heat stress condition.

Material and methods
The experimental methods regarding Animal Care were approved by the Animal Ethics Committee of the Bu-Ali Sina University in Iran.
Feed and water were offered ad libitum.Light was provided for 24 hour day -1 and gradually was reduced to 23 hour day -1 , and temperature was gradually reduced 3°C from the initial 32°C in each week.Feed intake (FI) and body weight gain (BWG) were measured weekly.Thus feed conversion ratio (FCR) was calculated.

Carcass traits
On 42 days of age, two birds from each replicate were randomLy selected, tagged, and weighted, then fasted for 8 hours (no limitation of water access) and were slaughtered.Carcass weight was measured after removal of feather, head, legs and abdominal contents.The abdominal fat dissected and recorded.The breast and thigh weights were calculated as the percentage of live body weights.

Blood parameters
On 42 days of age, two chickens selected randomLy from each replicate (n = 3) and blood taken from their wing vein and then immediately transferred on ice to laboratory.Serum samples were taken (centrifuged 3,500 × rpm for 15 min) and blood elements (Na, K, and Cl) were measured by ion selective electrode method of electrolyte analysis system (Kartium, Model E921-XI).In addition, one extra chicken was selected from each replicate and 2 (for blood gases assay) and 1 mL (for blood cell count) blood were taken from their wing vein and transferred into heparin tubes.Blood cell counts, hemoglobin and hematocrit (Hk) was measured using cell counter (Model, ERMA-Japan).In addition, Gimsa stain was used to heterophile (H), and lymphocytes (L) measurement.Moreover, blood gas (CO 2 , O 2 , and HCO 3 ) concentrations were determined using 9950IVL system (Switzerland).

Statistical analysis
All data were analyzed with CP levels, AA levels, and DEB as factorial 2 × 2 × 3 using a completely randomized design by the GLM procedure of Statistical Analysis System (SAS, 2004).Significant differences were compared by Duncan's multiple range test (p ≤ 0.05).

Results
The effects of experimental diets on broiler performance at grower (13-26 days of age) and finisher (27-42 days of age) periods are presented in Tables 3 and 4, respectively.The 21% CP level increased BWG and improved FCR at grower period (p < 0.05).In contrast, BWG decreased and FCR increased by 20% CP at finisher period (p < 0.05).Other parameters did not affected by AA and DEB levels (p < 0.05) (Table 4).The effects of treatments on carcass traits of broiler chickens at 42 days are presented in Table 5.The results indicated that the level of 20% CP increased carcass percentage (p < 0.05) but other parameters or traits were not affected by AA and DEB levels.The results of diets on blood parameters of broiler chickens at 42 days of age are presented in Table 6.The results demonstrated that 20% CP level was significantly increased Hb concentration than 17% CP level (p < 0.05).Hematological profile of blood is good indicator of stress in birds.The results of blood gas concentration of broiler chickens in response to diets at 42 days of age are given in Table 7.The results indicated that CP, AA, and DEB levels had no significantly effects on blood pH, P O2 , P CO2 , and HCO 3 concentrations.Increased respiration rates induced by severe heat stress seemed necessary for evaporative cooling in broilers.The results of blood ingredient concentration of broiler chickens at 42 days of age are presented in Table 8.The results have shown that 20% CP level significantly reduced Na concentration and electrolyte balance than 17% CP level (p < 0.05).The highest electrolyte balance was achieved by the level of DEB 320 mEq kg -1 other than treatments (p < 0.05).Means with different superscripts in each part of each same column are different (p < 0.05), μ±SD: Means±standard deviation, SEM = Standard error of the means, 1 Crude Protein; 2 Amino Acid; 3 According to Ross 308 International recommendation (Aviagen, 2013), AA standard (Met: 0.181, Thr: 0.030, and Lys: 0.149) for 17% CP and AA standard (Met: 0.112, Thr: 0.000, and Lys: 0.000) for 20% CP; 4 AA additional (Met: 0.214, Thr: 0.060, and Lys: 0.213) for 17% CP and AA additional (Met: 0.147, Thr: 0.000, and Lys: 0.000) for 20% CP; 5 Diet Electrolyte Balanc.

Discussion
By using modern broiler chickens, the suitable DEB for starter and grower period reported 246-315 and 249-257 mEq kg -1 , respectively (Murakami et al., 2001) which are in contrast with results of current study.The results are in agreement with the findings of Temim et al. (1999), who reported that broilers feeding with high protein diets (25 vs. 20%) had higher BWG during grower period under heat stress condition (32°C).Because of the different levels of protein and strain of broiler, changes in nutrition requirements and metabolism, similar results did not observe in finisher period.Balnave and Oliva (1991) reported that maximum BWG and FI observed at 30°C with DEB 380 mEq kg -1 .Under moderate environmental condition, However, the effects of different DEB (87, 180, 280 and 380 mEq kg -1 ) treatments were non-significant.Borges, Silva, Maiorka, Hooge, and Cummings (2004) reported that DEB of 240 was most favorable for broilers in either temperature environment (22.5±3.5°C; and cyclic stress 22.5±3.5°Cfor 14 hours and 33±2.0°C for 10 hours).Borges, Silva, Ariki, Hooge, & Cummings (2003a) reported that FI linearly increase with increasing of DEB (40-340 mEq kg -1 ).Conflicting findings emphasized that the effects of DEB on broiler performance is diverse and seems depends on differences in basal diet, broiler strain, temperature of rearing and supplemented electrolyte salts.Moreover, increasing of CP levels with essential AA in 32°C temperature increased FI in male broiler chickens (Alleman & Leclercq, 1997) that is in contrast with present study.This inconsistency could be related to differences in amount of CP levels and severity of heat stress condition.On the other hand, Flemming et al. (2001) compared three different levels of high, modulate, and low DEB in broiler chickens during the summer, and indicated that DEB had no effect on BWG.In addition, Borges et al. (2003a) pointed out that diets with DEB 240 mEq kg -1 was showed the best BWG than diets with DEB 0, 120, and 360 mEq kg -1 in broiler chicken at 23-30ºC.In this study, using 21% CP level in grower period and 17% CP level in finisher period had significant improvement on FCR than 19 and 20% CP levels in grower and finisher periods, respectively (p < 0.05).The results of present study are match with findings of Flemmnig et al. (2001) that FCR did not affect by various DEB levels.However, Borges et al. (2003a) showed that 240 mEq kg -1 DEB had best FCR comes before DEB than 120 and 360 mEq kg -1 in broiler chickens under 30-32°C.The results showed that different CP, AA, DEB levels, and their interaction had not significantly effect on mortality.Results are in agreement of other researchers (Flemming et al., 2001;Borges et al., 2003a), which no significant mortality was found by different DEB in broiler chickens under various temperature condition.Kamran and Mirza (2004) observed that broiler carcass percentage significantly increased by fed diet with 20% CP in compared to 21, 22, and 23% CP levels.These results are in contrast with present study.No significant differences were obtained in other traits.Ahmad and Sarwar (2005) showed that different sources of DEB supplemented with sodium bicarbonate increased significantly carcass weights, which supported the finding of current study.It is reported that DEB 40-340 mEq kg -1 had no effects on abdominal fat and carcass percentage (Borges et al., 2003a) which are inconsistence with the results of present study.Different experimental period (grower period) and severity of heat stress could explain to some extent of this inconsistency.Moreover, the similar breast and thighs percentage were reported in broiler chickens fed diets supplemented with Lys at 15-49 days of age (Holsheimer & Ruesink, 1993).
The differential leukocyte count and the monitoring of its changes in relation to effects of heat stress are an important part of the hematological profile of broilers (Ahmad & Sarwar, 2005).In contrast with the results of present study, the finding of Safamehr, Narimani, and Nobakht (2012) indicated that different levels of CP had no significant effect on broiler blood Hb concentration.Furthermore, 320 mEq kg -1 level DEB increased Hk percentage than 220 mEq kg -1 level but decreased H to L ratio (p < 0.05).No significant differences were observed on other parameters.Water loss by evaporation through respiration in heat stress induced an increase in plasma volume (hemodilution), that caused blood Hk and Hb values to be lowered down.Any therapy that increases blood Hk and Hb values in heat stress will be beneficial (Ahmad & Sarwar 2005).Therefore, increasing DEB up to 320 mEq kg -1 level could help to bearing of heat stress to broilers.The H and H to L ratio have been indicated to be reliable indicator to measure of stress (Zulkifli & Siegel, 1995).Under heat stress the H increase and L decrease.Maxwell and Robertson (1998) pointed out that moderate exposures to most stressors caused an increase in the number of H, and the H to L ratio in chickens.In contrast, Borges, Silva, Ariki, Hooge, and Cummings (2003b) reported significantly low H to L ratio for DEB 240 than those of 40, 140 and 340 DEB in both thermoneutral and heat stressed rooms.The results of current study are in contrast with the finding of Safamehr et al. (2012).These could be due to differences in DEB levels, intensity of heat stress, or bird., Strakova, & Suchy, 2003).This increase in Hk concentration is due to DEB increases.During severe panting, decreased blood partial pressure of CO 2 and, blood HCO 3 concentration but increased pH resulted in respiratory alkalosis and blood acid -base perturbations (Teeter, Smith, & Owens, 1985;Raup & Bottje, 1990).Present results indicated that during panting phase, blood pH was not as much as worse by treatments and it remained relatively constant in the range of 7.38 to 7.44.In contrast to the present findings, Teeter, Smith, and Owens (1985) reported that blood pH was elevated by chronic heat stress (32°C), and broilers had intermittent respiratory alkalosis during panting.
It is reported that broiler chickens fed diets with DEB 360 mEq kg -1 increased blood pH, and HCO 3 rather DEB 0, 120, 240 mEq kg -1 without any significant effects on blood P CO2 and P O2 under relatively heat stress and relative moisture condition at 42 days of age (Borges et al,. 2003a).The results of blood CO 2 is in agreement but the results of blood pH, P O2, and HCO 3 -are in contrast with the findings Borges et al. (2003a).Moreover, DEB 360 mEq kg -1 increased pH and blood HCO 3 rather 0, 120 and 240 mEq kg -1 without any affection on blood P CO2 and this inconsistency could be due to variation in the type of broiler chickens and severity of heat stress.Consequently, at high temperature, birds increase their respiration rate in an attempt to increase the rate of evaporative cooling.Such hyperventilation increases CO 2 losses and as a result respiratory alkalosis develops (Belay & Teeter, 1993) increase pH blood, and thereby increase blood CO 2 as well as decrease blood HCO 3 (Borges et al., 2003a).Khone and Jones (1975) reported that an increase in blood K level in response to heat stress.This K response seems to be related to the time which birds are under stress.It is reported DEB affected Cl -levels under heat stress as serum Cl -increased with increasing DEB levels (Ahmad and Sarwar, 2005).These observations are opposite of the results of present study which could be because of different DEB and age of broiler chickens.No significant difference were observed regarding Cl, Na, and K levels of blood serum with increasing DEB from 40 to 340 mEq kg -1 (Borges et al., 2003a).These results confirmed by the results of present study at finisher period.Moreover, in opposite of current study the different CP levels significantly changed Na on blood elements (Safamehr et al., 2009).In addition, DEB at levels 147 to 247 mEq kg -1 and calcium carbonate supplementation increased in blood electrolyte balance concentration (Olanrewaju et al., 2006) that is in according to results of present study.

Conclusion
Under heat stress challenges, broiler chickens used compensatory mechanisms when attempting to maintain acid-base homeostasis and concentrations of blood nutrients and physiological variables.These reactions make difficult to explain more precisely interaction of CP, AA, and DEB in the heat stress condition.However, an identical DEB 320 mEq kg -1 with 21% CP at grower and 17% CP at finisher period gave better carcasses traits and performance parameters with more reduction of sever of heat stress (H to L ratio).The present study indicates that AA levels had no obvious interaction with DEB and CP levels.

Table 2 .
Composition of experimental diets at finisher period (27-42 days of age, % or as is).

Table 3 .
The effect of treatments on broiler performance at grower (13-26 days of age).

Table 4 .
The effect of treatments on broiler performance (27-42 days of age).

Table 5 .
The effects of dietary treatments on carcass traits (% of × live body weight) and abdominal fat (% of body weight) at 42 days of age.

Table 6 .
The effect of treatments on blood parameters of broilers at 42 days of age.

Table 7 .
Blood gas concentrations (mm hg) of broiler chickens in response to diets at 42 days of age.

Table 8 .
The effect of diets on blood constituents of broiler chickens at 42 days of age (mmoL L -1 or mentioned).