Pulmonary hypertension and ascites as affected by dietary protein source in broiler chickens reared in cool temperature at high altitudes

Animal Feed Science and Technology 155 (2010) 194–200

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Pulmonary hypertension and ascites as affected by dietary protein source in broiler chickens reared in cool temperature at high altitudes M. Izadinia a , M. Nobakht a , F. Khajali a,∗ , M. Faraji b , F. Zamani b , D. Qujeq c , I. Karimi d a b c d

Department of Animal Science, Shahrekord University, Shahrekord, Iran Agricultural Research Center, Shahrekord, Iran Department of Biochemistry and Biophysics, Babol University for Medical Sciences, Babol, Iran Department of Clinical Sciences, Shahrekord University, Shahrekord, Iran

a r t i c l e

i n f o

Article history: Received 2 March 2009 Received in revised form 17 December 2009 Accepted 21 December 2009 Keywords: Broilers Canola meal Chronic heart failure Pulmonary hypertension Soybean meal

a b s t r a c t As canola meal protein (CMP) has a lower arginine (Arg) content than soybean meal protein (SBMP), the main objective of this study was to investigate if replacing SBMP by CMP affected the incidence of pulmonary hypertension and ascites. Two hundred and seventy broiler chicks (Ross 308) were used to evaluate the effect of replacing CMP at proportionally 0, 0.5, and 1 for SBMP in a complete randomized block design, as mentioned in the Statistical analysis section (dietary treatments designated as CMP0, CMP0.5, and CMP1, respectively). The experiment was carried out on litter flooring from 6 to 42 days of age. The results indicate that broiler performance (body weight gain, feed intake, and feed conversion ratio) was significantly (P<0.05) impaired in the CMP1 group at 6–21 days but not at 21–42 days. Inclusion of canola meal (CM) in the diet significantly increased ascites and total mortality (P<0.05) and caused a decrease in the serum nitric oxide (NO) concentration with a concomitant increase (P<0.05) in the right ventricular weight ratio (RV/TV). Haematocrit, circulatory triiodothyronine and thyroxine thyroid hormone concentrations and serum alkaline phosphatase activity were unaffected by dietary treatments. In conclusion, the results of this study indicate that substitution of CMP for SBMP was associated with pulmonary hypertension (i.e., higher RV/TV) and ascites mortality in broiler chickens. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Canola meal (CM) is characterized as having a low apparent metabolizable energy (AME) content. There is variation in AME content among CM prepared from different canola cultivars. Among the commercial meals, the yellow-seeded canola had a significantly higher AME value than the conventional sample from brown-seeded canola (Slominski et al., 1999). CM has lower amino acid digestibility values than SBM but the reasons are not well known. Although CMP is rich in sulfur amino acids, arginine (Arg) content of this oilseed is approximately two-thirds that of SBMP (20.8 g/kg vs 31.4 g/kg according to NRC (1994)) as well as having a lower Arg digestibility compared to SBMP (Heartland Lysine, 1998). Substitution of a high proportion of CMP at the expense of SBMP in poultry diets may drop the dietary Arg level below its requirements. Dietary Arg levels advocated by NRC (1994) are minimal requirements established under conditions of

Abbreviations: CMP, canola meal protein; SBMP, soybean meal protein; Arg, arginine; RV/TV, right ventricle to total ventricles weight ratio; AME, apparent metabolizable energy; NO, nitric oxide. ∗ Corresponding author. Tel.: +98 381 442 4401; fax: +98 381 442 4428. E-mail addresses: [email protected], [email protected] (F. Khajali). 0377-8401/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2009.12.009

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thermoneutrality and optimal dietary and physiological conditions. However, NRC recommendations may not be adequate to support maximal growth, support Arg-depleting immune responses, and prevent the onset of pulmonary hypertension in broilers reared under suboptimal environmental conditions (Bowen et al., 2007; Wideman et al., 1996). Arg is an essential amino acid for birds because carbamyl phosphate synthetase I is absent in uricotelic species and thus cannot be synthesized in birds (Sung et al., 1991). Arg has been documented to be the precursor of NO, a potent vasodilator (Collier and Vallance, 1989). Thus, if CM is included at high levels in a broiler diet, it is possible that dietary Arg content may not be adequate to fully support the production of NO by avian macrophages and the pulmonary vascular endothelium (Wideman et al., 1996). Diminished NO availability and production have been implicated in the pathogenesis of pulmonary hypertension and in other vascular disorders including atherosclerosis (Shaul, 2002). Newkirk and Classen (2002) conducted an experiment in which SBM was replaced by CM. The results showed that total mortality and chronic heart failure (ascites) rates between 19 and 39 days increased with level of CM addition from 5.2% to 13.9% and 1.9% to 9.6%, respectively. The high rate of chronic heart failure in the chickens fed low-glucosinolate CM remained unexplained. As ascites is a common problem at high altitudes (Albers and Frankenhuis, 1990; Khajali et al., 2007), research needs to be done to evaluate the impact of such dietary ingredient substitution on productivity and ascites in broiler chickens. Atmospheric oxygen less than 19.6%, corresponding to approximately 1000 m above sea level (Julian, 2000), may increase the incidences of ascites mortality (Beker et al., 2003). Given that Arg requirements of broiler chickens are likely to be higher than NRC recommendations in extreme environmental conditions (high altitude and cool temperatures), the hypothesis tested in the present study was that substitution of SBMP with CMP will increase the incidence of pulmonary hypertension and ascites. 2. Materials and methods The experiment was conducted in Shahrekord (Iran), an area with the altitude of 2100 m above the sea level. The experimental animals were kept, maintained and treated in accepted standards for the humane treatment of animals. The Institutional Animal Care and Use Committee of Shahrekord University approved all procedures used in this study. 2.1. Experimental design Three different substitution levels of CMP for SBMP were used. Each substitution level was designated as a treatment. The experiment was conducted in a randomized block design. 2.2. Birds Day old male broiler chicks (Ross 308) were obtained from a local hatchery and placed in a house and raised for 6 days. After removing obvious runts, 270 day old broiler chickens were randomly assigned to one of 18 pens (15 birds/pen) with wood shaving litter. 2.3. Experimental diets and husbandry The canola meal was obtained from a local provider company located in northern Iran. The CMP replaced at proportionally 0, 0.5, and 1 for dietary SBMP. Dietary treatments are designated as CM0, CM0.5, and CM1, respectively, thereafter. Diets were formulated in a way to be isonitrogenous and isoenergetic and to meet bird requirements according to the recommendations of NRC (1994) in each feeding phase. Feeding phases were starter (0–3 wk of age) and grower (3–6 wk of age). Diets were presented in a crumble form. Table 1 presents the composition of diets in two feeding phases. Six litter pens were randomly allotted to each of three dietary treatments. Feed and water were provided ad libitum during the trial. A cool model was used to trigger ascites in the birds. The initial house temperature was 32 ◦ C and decreased in a stepwise fashion to 25 ◦ C by day 7, 20 ◦ C by day 14 and 15 ◦ C by day 21 at which it was maintained to the end (42 days) as applied previously (Khajali et al., 2007). The lighting program consisted of 23L:1D with intensity of 15 lx throughout the trial. 2.4. Measurements Body weight and feed consumption were recorded at the beginning and the end of the feeding phases and feed conversion ratio (FCR) was calculated taking into account the mortality weights in different stages of the experiment. Blood samples were collected from the brachial vein from two birds per pen (12 birds/treatment) at 42 days of age and centrifuged at 2500 × g for 10 min. Blood for haematocrit measurements was collected into heparinised microcapillary tubes and centrifuged in a microlitre centrifuge (BMC 24 Microhaematocrit centrifuge, Pars Azma Co., Isfahan, Iran) for 5 min. Sera were used for the determination of triiodothyronine (T3 ) and thyroxine (T4 ) levels with a commercial kit (KT2CT; Radim, Barcelona, Spain) as well as NO concentration and total alkaline phosphatase activity (ALP) (EC 3.1.3.1). Serum NO concentration was determined according to Hortelano et al. (1995) by adding 250 ␮l of serum to 1 ml of Griess reagent. The Griess reagent was

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Table 1 Composition of the experimental diets (g/kg) (as fed). Feed ingredient

Amount in the starter diet (g/kg)

Amount in the grower diet (g/kg)

CMP0

CMP0.5

CMP1

CMP0

CMP0.5

CMP1

Maize Soya bean meal (44% CP) Canola meal (34%) Fish meal (60% CP) Soya refined oil Wheat bran Dicalcium phosphate Oyster shell Salt Trace mineral premixa Vitamin premixb dl-methionine l-HCl–lysine AME (MJ/kg)

569.5 341 – 25 27 – 13 14 4.0 2.5 2.5 1.5 – 12.55

513 160 207 50 41 – 8.5 11 4.0 2.5 2.5 0.5 – 12.55

421 – 435 50 65 4.0 8.0 8.0 4.0 2.5 2.5 – – 12.55

645 301 – – 18.5 – 12 14 3.0 2.5 2.5 1.5 – 12.55

570 153 205 – 38 1.5 11 12 4.0 2.5 2.5 – 0.5 12.55

544 – 353.4 35 42 – 10 7.5 0.3 2.5 2.5 – – 12.55

Analyzed values CP (g/kg) Met + cys (g/kg) Lys (g/kg) Arg (g/kg) Thr (g/kg) Crude fiber (g/kg) Ether extract (g/kg)

214 8.5(9.0) 11.3(11.8) 13.0(14.7) 7.9(8.2) 22 60

215 8.5(9.0) 11.7(11.8) 12.6(13.2) 8.3(8.6) 41 67

214 9.0(9.0) 11.6(11.8) 12.0(12.6) 8.5(9.0) 61 80

187 6.5(8.1) 9.5(9.9) 11.5(11.9) 6.9(7.1) 22 51

186 6.7(8.1) 9.0(9.9) 10.9(11.2) 7.1(7.4) 40 60

187 6.9(8.1) 9.6(9.9) 10.4(10.8) 7.3(7.6) 54 62

Calculated analysis Ca (g/kg) Available P (g/kg) Na (g/kg) K (g/kg) Cl (g/kg) Na + K−Cl (meq/kg)

9.5 4.4 2.0 8.5 2.8 226

9.5 4.4 2.0 7.5 2.9 196

9.5 4.4 2.0 6.5 2.8 175

8.5 3.4 1.4 8.0 2.2 204

8.5 3.4 1.4 7.0 2.2 179

8.5 3.4 1.4 6.0 2.1 156

5.6

11.7

5.5

9.6

Total glucosinolates (␮mol/g)

0

0

Values in parenthesis are calculated amino acids. a Provided the following per kg of diet: Mn (from MnSO4 ·H2 O), 40 mg; Zn (from ZnO), 40 mg; Fe (from FeSO4 ·7H2 O), 20 mg; Cu (from CuSO4 ·5H2 O), 4 mg; I (from Ca(IO3 )2 ·H2 O), 0.64 mg; Se (from sodium selenite), 0.08 mg. b Provided the following per kg of diet: vitamin A (trans-retinyl acetate), 3600 IU; vitamin D3 (cholecalciferol), 800 IU; vitamin E (dl-␣-tocopheryl acetate), 7.2 mg; vitamin K3 , 1.6 mg; vitamin B1 , 0.72 mg; vitamin B2 , 3.3 mg; vitamin B3 , 0.4 mg; vitamin B6 , 1.2 mg; vitamin B12 , 0.6 mg; folic acid, 0.5 mg; choline chloride, 200 mg.

a mixture (1:1) of 1% sulfanilamide in 5% phosphoric acid and 0.1% 1-naphthylethylenediamine giving a red-violet colour in presence of nitrite, the stable form of NO. The absorbance was measured at 540 nm by means of a spectrophotometer (Corning 480, USA). For the activity of serum ALP, the rate of formation of the yellow colour of p-nitrophenol produced by hydrolysis of p-nitrophenylphosphate in alkaline solution was measured colourimetrically according to Vertommen et al. (1980). 0.1 ml of serum was incubated for 30 min in a buffer (pH 9.3) containing 5 mmol glycine, 0.05 mmol MgCl2 and 5 mmol p-nitrophenylphosphate per liter. The reaction was stopped by addition of 10 ml of 0.02N sodium hydroxide. The colour intensity was read at 405 nm. The enzyme activity was expressed as U where a unit is the amount of enzyme which catalyses the liberation of 1 ␮mol nitrophenol per liter of serum per minute. All chemical reagents were obtained from Sigma–Aldrich Co. (Sigma–Aldrich Co., St. Louis, MO, USA). All floor pens were checked daily for mortality and dead birds were necropsied for the recognition of the cause of death. Visually, birds with ascites exhibit characteristic dark red combs indicating cyanosis and they are also reluctant to move. Post-mortem examinations including accumulation of fluid in the abdominal cavity and the ratio of right ventricle to total ventricles (RV/TV) were performed to confirm the ascites (Khajali and Fahimi, 2010). The RV/TV ratios that exceeded 0.299 were considered as ascites (Walton et al., 2001). At the end of the experiment (day 42), 20 birds from each treatment were decapitated for the determination of RV/TV as a measure of pulmonary hypertension. 2.5. Chemical analysis Chemical composition of feed ingredients and experimental diets for dry matter (DM), crude protein (CP), crude fibre (CF) and crude fat (EE) were determined by Association of Official Analytical Chemists (AOAC) methods 934.01, 992.93, 962.09 and 920.39, respectively (AOAC, 2000). Amino acid composition of the experimental diets was also determined. To determine amino acids, duplicate samples of each diet were subjected to 6N HCl and hydrolyzed for 24 h at 110 ◦ C (Andrew and Baldar,

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Table 2 Weight gain, feed intake and feed conversion ratio of broilers that received different sources of dietary protein during the experiment.a . Variables

Feed intake (g/b) 6–21 days 21–42 days 6–42 days

Diets

SEM

P-value

CMP0

CMP0.5

CMP1

759a 2852 3612a

749a 2725 3474ab

721b 2698 3423b

2.5 25.7 22.0

0.001 0.101 0.020

425b 1324 1749

1.8 12.7 12.8

<0.001 0.732 0.254

Body weight gain (g/b) 6–21 days 21–42 days 6–42 days Feed conversion ratio 6–21 days 21–42 days 6–42 days

467a 1359 1826 1.62b 2.10 1.97

463a 1334 1797 1.62b 2.05 1.94

1.69b 2.05 1.96

0.007 0.023 0.018

0.010 0.462 0.651

Means in the same row with different letters (a and b) are significantly different. a Each mean represents values from six replicates.

1985). After acid hydrolysis, all samples were analyzed for amino acid content by use of an ion-exchange chromatograph (LKB Biochrom 4141, UK). Performic acid oxidation was done to determine sulfur amino acids (Moore and Stain, 1963). 2.6. Statistical analysis Data from the experiment were analyzed in a complete randomized block design using JMP software (SAS Inst. Inc., Cary, NY). Duncan’s multiple range test was used to compare and separate treatment means. The statistical model was Yij =  + Ti + Bj + D ij , where Yij is observation;  is the general location parameter (i.e., the mean); Ti is the effect for being in treatment i; Bj is the effect for being in block j; and D ij is random error. The differences between the means were significant at P≤0.05. 3. Results Analyzed and calculated values of amino acid contents of the experimental diets were in close agreement (Table 1). Dietary Arg level decreased by increasing the level of CM in diets as no synthetic Arg supplement was used. Arg levels of CMP0.5 and CMP1 were marginally adequate (12 and 10.4 g/kg in the starter and grower periods vs 11.7 and 10 g/kg) recommended levels by NRC (1994) to support optimal performance. 3.1. Growth performance Feeding CMP1 caused a significant reduction in weight gain (P<0.001) and feed intake (P=0.001) and resulted in impaired FCR (P=0.01) during 6–21 days (Table 2). The birds fed CMP1 had 9% and 5% less feed consumption and weight gain, respectively, than those fed CMP0. FCR was depressed (P=0.01) by the addition of CMP0 in the diet by 7 units (1.69 vs 1.62). However, the birds fed CMP0.5 had feed intake and weight gain comparable to those fed 0 with the same FCR. During 21–42 days, no significant difference was observed with regard to weight gain, feed intake and FCR among the treatments. A significant difference was found between CMP0 and CMP1 in terms of feed intake for the entire study (6–42 days) (P<0.05). However, the differences between these treatments with respect to body weight gain and FCR were not significant (Table 2). 3.2. Physiological variables Table 3 depicts physiological variables measured at the end of experiment (42 days) in broilers receiving different sources of dietary protein. Haematocrit was not significantly increased by the dietary treatments. Serum NO concentrations declined with increasing inclusion of CM in broiler diets so that the difference between CMP0 and CMP1 was significant (P<0.05). Circulatory thyroid hormones were not affected by dietary protein source. There was no significant change in the activity of serum alkaline phosphatase by replacing CM in the diet. The ratio of RV/TV showed a significant elevation as CM included in the diet (Table 3). A significant rise in total mortality and mortality from ascites was observed by substituting SBMP with CMP in the diet. Total mortality and mortality from ascites were significantly (P<0.05) greater in birds fed CMP1 than those fed CM0. Birds fed CMP0 had the lowest level of mortality whether or not related to ascites (Table 3). Almost all mortality occurred toward the end of the growing period.

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Table 3 Variables assessed at 42 days of age in broilers that received different sources of dietary protein. Variables

Haematocrita (%) Serum nitric oxidea (␮mol) Serum triiodothyroninea (T3 ) (ng/ml) Serum thyroxinea (T4 ) (␮g/dl) Alkaline phosphatasea (U)b RV/TVc Total mortality (%) Ascites mortality (%)

Diets CMP0

CMP0.5

CMP1

38.8 5.94a 1.81 1.14 402 0.21c 5.0b 3.4b

38.8 5.61ab 2.03 1.02 431 0.25b 8.8b 6.7ab

39.8 5.15b 1.76 1.04 485 0.27a 21.1a 15.0a

SEM

P-value

0.21 0.052 0.033 0.017 17.4 0.001 1.12 0.77

0.724 0.008 0.191 0.284 0.592 <0.001 0.033 0.031

Means in the same row with different letters (a, b, and c) are significantly different. RV/TV, right ventricle to total ventricles. a Each mean represents values from 12 replicates. b One unit is the amount of enzyme which catalyses the liberation of 1 ␮mol nitrophenol per liter per minute. c Each mean represents values from 20 replicates.

4. Discussion 4.1. Growth performance In general, broiler performance was low in the present trial because of high altitude as well as cold exposure. The partial pressure of oxygen drops approximately 7 mm Hg for each 1000 m increase in altitude, reducing the amount of oxygen available to the hemoglobin in red blood cells as blood passes through the lungs. This is equivalent to a drop of approximately 2.5% in the air oxygen for every 1000 m increase in altitude (Julian, 2007). In other words, each 500 m increase in altitude above sea level reduces oxygen availability by about 1% from 20.95% at sea level (Julian, 2007). The significant reduction observed in broiler performance (1–21 days) as a consequence of feeding CMP at the expense of SBMP can be explained by a number of reasons, as follows. Nutrient requirements advocated by NRC (1994) have been established under thermoneutral conditions and they are minimal requirements. There are several reports indicating that Arg requirements of broilers are influenced by ambient temperature (Chamruspollert et al., 2004). Brake et al. (1998) reported that during hot weather Arg requirements are increased, and they advised Arg supplementation under heat stress conditions. The same situation can happen in cold stress conditions. Likewise, the hypobaric condition of high altitude is a stressful factor in broiler production. In the present study, the birds were raised at cool temperature and at a severely hypoxic condition. Under these conditions cardiac output is increased to deliver more oxygen to the tissues. The elevated pulmonary arterial pressure (as speculated by significant increase in RV/TV) causes a substantial increase in NO production by the pulmonary vascular endothelium. To support increased NO production, Arg requirements will increase when broilers are raised under these rigorous conditions. It should be realized that the pulmonary vascular capacity of modern broilers is marginally adequate to accommodate the cardiac output required to support the metabolic demands incurred by fast growth and the extremes of environmental temperatures (Wideman et al., 2007). Nevertheless, there are reports suggesting Arg supplementation beyond the NRC recommendations improves broiler productivity (Fernandez et al., 2009; Ruiz-Feria, 2009). Kidd et al. (2001) reported that supplementing broiler diets with 2 g/kg Arg beyond NRC requirements even under normal conditions resulted in improved growth performance. CM has a relatively high glucosinolates (Gls) content that may limit its efficient utilization. Glucosinolates exert deleterious effects on all classes of animals including poultry. Severe growth depression and increased mortality in broiler chickens are attributable to a total Gls content above 8.0 ␮mol/g diet (Tripathi and Mirsha, 2007). Mawson et al. (1994) indicated that when the level of Gls increased to 6–10 ␮mol/g there was a mild reduction in growth, and when exceeding 10 ␮mol/g the growth was severely affected. In the present study, dietary levels of Gls in CMP1 in the staring period were greater than 10 ␮mol/g and it might have affected the performance of chickens. Dietary electrolyte balance (DEB) tended to decrease by replacing CMP for SBMP (Table 1). However, it is unlikely that such a change in DEB can affect the growth performance of broiler chickens. It has been shown that different DEB ranging from 87 to 380 mEq/kg did not influence the broiler performance (Balnave and Oliva, 1991). Hulan et al. (1987) observed little difference in the performance of chicks fed on diets in which DEB varied from 155 to 300 mEq/kg. Borges et al. (2003) reported an optimal DEB of 240 mEq/kg for maximum weight gain and 207 mEq/kg for optimum FCR in thermoneutral and hot environments. 4.2. Physiological variables Haematocrit values reported herein are higher than those reported at low altitudes. Blood haematocrit is a sensitive indicator of physiological change attributable to atmospheric oxygen. Beker et al. (2003) suggested that 19.6% atmospheric O2 is the minimal allowable level for housing birds within a relatively stress-free environment to avoid cardiac and haematocrit changes related to ascites. The availability of atmospheric oxygen is estimated to be 17% in this study and this is the reason

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that haematocrit values are very high. Our finding is consistent with the report of Ruiz-Feria (2009) who indicated that supplementing 10 g/kg Arg to commercial broiler diets resulted in a significant reduction in the haematocrit. The right ventricular weight ratio (RV:TV) is an index of pulmonary hypertension. The ratios that exceed 0.25 manifest pulmonary hypertension in chickens (Squires and Julian, 2001). Birds subjected to CM diets had a significantly higher RV/TV ratio indicating pulmonary hypertension. The cascade of events leading to such a condition is triggered by hypoxemia. Pulmonary hypertension causes the right side of the heart to pump more blood to the lung and as a result the wall of the right ventricle is thickened. Newkirk and Classen (2002) reported that feeding CM resulted in a linear increase in heart weight as a proportion of the body weight, which may be a sign of chronic heart failure (ascites). Complete substitution of CMP for SBMP caused a significant reduction in serum NO concentration. In fact, Arg serves as precursor for NO and the lower content of Arg in CM group resulted in a lower serum NO level. NO, previously referred to as relaxing factor, is responsible for vasodilatation that is essential for the regulation of blood pressure (Shaul, 2002). The pulmonary arterial relaxation was improved when Arg was supplemented above the NRC (1994) requirements, and this has been attributed to an increase in NO production (Wideman et al., 1996). Ruiz-Feria (2009) found that supplementation of Arg in combination with antioxidant vitamins to broiler diets significantly increased the plasma concentrations of NO in the plasma. The RV:TV and serum circulatory NO values in the present experiments matched the results of ascites mortality, further supporting the role of dietary protein source on pulmonary hypertension and ascites. By considering total mortality and mortality from ascites, it is evident that ascites was the major cause of death in broiler chickens under conditions of the trial and that the substitution of CMP at the expense of SBMP caused higher mortality in broiler chickens. Mushtaq et al. (2007) reported that inclusion of CM at 300 g/kg of broiler diets caused higher mortality but their study was not related to ascites. In the study conducted by Newkirk and Classen (2002), total and ascitic mortality of broilers increased from 5.2% to 13.9% and 1.9% to 9.6%, respectively, by replacing soybean meal with CM between 19 and 39 days of age. This study used a low-glucosinolate CM and was conducted under thermoneutral condition in a low land area. 5. Conclusion In summary, dietary protein composition that cause marginally adequate levels of Arg for broilers grown at high altitudes, would not support the ascites-related variables. Complete substitution of CMP for SBMP significantly reduced feed intake of broiler chickens grown at a high altitude with a concomitant decrease in serum NO concentration and a significant increase in RV/TV and mortality from ascites. In addition, the substitution of CMP for SBMP caused mortality other than ascites. In conclusion, dietary protein source was associated with pulmonary hypertension and ascites in broiler chickens reared under conditions of a cool environment and high altitude. Acknowledgements Authors would gratefully thank Dr. Walter Bottje from the Center of Excellence for Poultry Science at University of Arkansas, Fayetteville, AR, USA, for editing the manuscript. This research was funded by Shahrekord University, Shahrekord, Iran. References Albers, G., Frankenhuis, A., 1990. Ascites, a high altitude disease in lowlands. Poult. Misset 6, 22–24. Andrew, R.P., Baldar, N.A., 1985. Amino acid analysis of feed constituents. Sci. Tools 32, 44–48. AOAC, 2000. 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