METABOLISM AND NUTRITION. Sodium and Chloride Requirements of Growing Broiler Chickens (Twenty-one to Forty-two Days of Age) Fed Corn-Soybean Diets

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1 METABOLISM AND NUTRITION Sodium and Chloride Requirements of Growing Broiler Chickens (Twenty-one to Forty-two Days of Age) Fed Corn-Soybean Diets A. E. Murakami, 1 E. O. Oviedo-Rondón, 2 E. N. Martins, M. S. Pereira, and C. Scapinello Departamento de Zootecnia, Universidade Estadual de Maringá, Avenida Colombo, Maringá, Paraná, , Brazil ABSTRACT Two trials were conducted to determine Na + levels. In the second trial, the Cl requirement was Na + and Cl nutritional requirements and dietary electrolyte balance (DEB) and its effects on acid-base balance, litter moisture, and incidence of tibial dyschondroplasia (TD) in broiler chickens during the growing period. Cobb broilers were distributed in a completely randomized design (30 pens) with six treatments, five replicates, and 50 birds per experimental unit at 21 d of age. Treatments used in both trials were a basal diet with 0.10% Na + (Trial 1) or Cl (Trial 2) supplemented to result in diets with Na + or Cl levels of 0.10, 0.15, 0.20, 0.25, 0.30, and 0.35%. In the first trial, the results indicated an optimum Na + requirement of 0.15%. The Na + levels, obtained with supplemental NaHCO 3, did not affect blood gas parameters and TD incidence. Litter moisture increased linearly with estimated at 0.23%. Increasing Cl levels, provided by NaCl with NaHCO 3 to balance Na +, caused a linear effect (P 0.01) on blood gas parameters, with an estimated equilibrium at 0.19% dietary Cl. No effect (P 0.05) of Cl levels on litter moisture was observed. The hypertrophic area of growth plate in the proximal tibiotarsus increased with Cl levels (P 0.001). A nonlinear model describes this response. The best dietary electrolyte balance (DEB) was between 250 to 261 meq/kg in Trial 1 and 249 to 257 meq/kg in Trial 2. We concluded that the Na + requirement was 0.15%, and the Cl requirement was 0.23% for maximum performance of growing chickens between 21 and 42 d of age, and the best DEB was between 249 and 261 meq/kg. (Key words: acid-base balance, chloride requirements, sodium, tibial dyschondroplasia) 2001 Poultry Science 80: INTRODUCTION Sodium (Na + ) and Cl are low-cost nutrients, and their manipulation has little influence on the diet cost. However, because of their important metabolic effects on nerve cells, acid-base balance, osmotic pressure regulation, and monosaccharide and amino acid absorption, it is necessary to supply them in precise levels and adequate balance for optimum growth, bone development, and good litter quality. Researchers have expressed differences of opinion regarding the most appropriate requirement values. The NRC (1994) established a recommended requirement of 0.15% each for Na + and Cl for growing chickens. Zanardo (1994) estimated a requirement of 0.20% Na + for the best chicken performance from 21 to 49 d of age. Murakami et al. (1997) determined a Na + requirement of 0.15% and not more than 0.20% of Cl from 42 to 56 d of age. The same authors reported no difference Received for publication December 27, Accepted for publication October 26, To whom correspondence should be addressed: aemurakami@ uem.br. 2 Present address: Department of Poultry Science, University of Arkansas, Fayetteville, AR between NaHCO 3 and NaCl as Na + sources. Barros et al. (1998) estimated a requirement of Na + of 0.25% for maximum body weight gain (BWG) and 0.29% for the best feed conversion ratio (FCR). Little research has been directed toward estimation of the Cl requirement for growing broilers. Because of physiological interactions of Na + and Cl with K +, the relationship between these ions have been evaluated in poultry diets (Dewar and Whitehead, 1973; Hurwitz et al., 1973; Ross, 1979; Johnson and Karunajeewa, 1985; Halley et al., 1987; Hooge, 1995, 1998). The ratios of Na +,Cl, and K + in the diet are important to determine dietary electrolyte balance (DEB) (Mongin, 1980; Teeter, 1997). The DEB is a principal factor in the acid-base balance regulation, which determines blood ph for better enzymatic efficiency and, thus, influences bird growth and performance (Patience, 1990; Butcher and Miles, 1994; Hooge, 1998). Mongin (1980) emphasized the importance of adjusting DEB to obtain optimum performance, because when the balance is altered due to acidosis or alkalosis, metabolic pathways cannot function with maximum ef- Abbreviation Key: BE = blood base excess; BWG = body weight gain; DEB = dietary electrolyte balance; FCR = feed conversion ratio; FI = feed intake; SO = saturation with O 2 ;TD= tibial dyschondroplasia. 289

2 290 MURAKAMI ET AL. TABLE 1. Composition of experimental diets 1 Calculated sodium levels (%) (Experiment 1) 2 Calculated chloride levels (%) (Experiment 2) 2 Ingredients Ground yellow corn Soybean meal Dicalcium phosphate Limestone Soybean oil DL-Methionine 99% Premix 4, Antioxidant (BHT) Sodium chloride NaHCO KCl K 2 SO Calculated analysis: Mongin no., 6 meq/kg All diets were calculated to contain 20.3% CP; 3,200 kcal/kg ME; 0.90% calcium; 0.35% available phosphorus; and in Experiment 1: 0.93% potassium, and in Experiment 2: 0.96% potassium. In Experiment 1, Cl was 0.15%, and in Experiment 2 Na + was 0.15%. 2 The determined levels of sodium, chloride, and potassium closely approximated calculated levels. 3 NRC (1994) control treatment. 4 Composition of vitamin premix (per kilogram of premix): vitamin A, 1,600,000 IU; cholecalciferol, 320,000 IU; vitamin E, 3,600 IU; menadione sodium bisulphite, 480 mg; vitamin B 1, 320 mg; vitamin B 2, 960 mg; vitamin B 6, 640 mg; folic acid, 160 mg; nicotinic acid, 4,800 mg; biotine, 16 mg; calcium pantothenate, 1,920 mg; choline chloride, 52,000 mg; vitamin B 12, 2,400 mg; antioxidant (BHT), 19,600 mg; coccidiostat (monensin), 120,000 mg; and growth promotor (zinc bacitracin), 20,000 mg. Supplied by Roche (São Paulo, Brazil). 5 Composition of mineral premix provided as follows per kilogram of premix: iron, 1,000,000 mg; manganese, 16,000 mg; zinc, 100,000 mg; copper, 20,000 mg; selenium, 50 mg, cobalt, 2,000 mg; and iodine, 2,000 mg. Supplied by Roche (São Paulo, Brazil). 6 Mongin number = [Na +, meq/kg + K +, meq/kg] Cl meq/kg (Mongin, 1980). ficiency. Both Na + and K + have indirect alkalogenic effects on body fluids, and bicarbonate has a buffering effect. The Cl ion has an indirect acidogenic effect, and Cl should be maintained at or near requirement levels, because Cl excess can cause leg and articulation problems in broiler chickens (Leach and Nesheim, 1972; Adesina and Robbins, 1987; Ruíz-López et al., 1993; Hooge, 1998). Halley et al. (1987) observed a high correlation between TD incidence and acid-base imbalance, which indicated effects of dietary mineral manipulation on blood buffering capacity, and bone mineralization. Johnson and Karunajeewa (1985) determined that the best DEB is between 200 and 350 meq/kg, which is influenced by phosphorus, K +,Na +, and Cl. Electrolyte salts are responsible for alteration of fecal and litter moisture. Both Na + and K + cause increases in water excretion and increase litter management problems (Damron et al., 1986; Penz, 1988; Hooge, 1995). A few publications have been found on the requirements of these minerals as linked to effects on metabolism, bone development, and litter quality. There is still some confusion about the best dietary levels for these minerals for growing chickens. Thus, the aim of this work was to determine nutritional Na + and Cl requirements and the best DEB for growing broiler chickens to obtain optimum performance and to observe metabolic influences in acid-base balance, TD incidence, and litter moisture in relation to dietary Na + and Cl levels. MATERIALS AND METHODS Two trials were done, each using 1, d-old male Cobb chickens. Birds were housed in an open-sided house with sidewall curtains, with 50 birds per pen (5.1 m 2 ). Fresh wood shavings were used as litter. A continuous illumination program was used during first week and then 20 h light:4 h darkness until slaughter. Feed and tap water were provided for consumption ad libitum. Chickens were vaccinated against Marek s disease at the hatchery and against Newcastle and Gumboro diseases at8dofage. Diet chemical analyses were made according to procedures described by AOAC (1990). The Na + concentrations were determined by atomic absorption spectrophotometry, and Cl was measured by AgNO 3 titration and K + by flame photometry. A tap water sample was analyzed to establish the mineral composition of Na +,K +, and Cl. Results indicated a concentration of these minerals less than 0.5 mg/kg. Birds were weighed at the start (21 d) and the end (42 d) of each experiment. When any bird died or was removed, it was weighed and pen feed intake (FI) at that time was recorded, and these data were used to correct FCR. Average maximum and minimum environmental temperatures in the house were recorded daily. To determine acid-base balance status, eight 2-mL blood samples per treatment were taken by cardiac puncture anaerobically at 35 d of age. Measurements were taken on ph, partial pressure of CO 2 (pco 2 ), partial pressure of O 2 (po 2 ), bicarbonate (HCO 3 ), CO 2 tension (TCO 2 ), base excess (BE), and saturation of hemoglobin with oxygen (SO). Minimum 80% and maximum 98% SO were used as criteria for a satisfactory sample. Body temperature of 42 C and 10.5 g/dl hemoglobin were used for device calibration (Ruíz-López and Austic, 1993).

3 SODIUM AND CHLORIDE REQUIREMENTS FOR BROILERS 291 To evaluate TD incidence, two 42-d-old chickens per experimental unit (10 per treatment) were weighed and killed, and two methods were used: 1) right leg was used for classification by the scoring system of Edwards and Veltmann (1983), and 2) the left epiphysial tibiae were fixed in Bouin s solution for histological analysis (Bec ak, 1976). Decalcification was made with Haug solution, to avoid bone tissue hydrolysis, and tissues were infiltrated with paraffin. Sections were made with rotatary microtome at 5 µm and were stained with hematoxilin-eosin, for epiphysial disc zone observation and characterization of TD lesions. Three regions were considered: growth plate band, proliferating cartilage, and hypertrophic cartilage. Calcified cartilage was considered as the lower limit to determine the thick hypertrophic zone in TD diagnosis (Thorp et al., 1993). The data collected on TD, at gross dissection and after histological assessment, was not normally distributed, and the cardinal numbering of the categories does not imply that the distance between categories is in any sense regular (McCullagh and Neder, 1983). In consideration of this, we designed a method to measure tibia zones with software for image analysis (Image-Pro ). 3 For this process, 10 tibia histological plates per treatment were scanned. Three areas were measured: 1) growing cartilage (growth plate and proliferating cartilage zones), 2) hypertrophic zone, and 3) total area of epiphysis, including chondro-epiphysis. These data were analyzed by statistical processes of generalized linear models (GLIM ) 4 (Nelder and Wedderman, 1972). Litter moisture score was evaluated at 41 d, by using four subsamples per experimental unit. A score of 1 = dry litter in good condition, and 4 = very damp litter. Average value was used to indicate litter score. Experiment 1 The response to different dietary Na + levels in 21 to 42-d-old chicken males was evaluated. Birds received the same management and fed the same diet formulated to satisfy recommendations of NRC (1994) for the 1- to 21-d starter period. Initial mean bird weight was 828 ± 0.02 g. Average maximum and minimum temperatures were 29 ± 2.5 C and 21 ± 1.5 C. Treatments consisted of a basal diet deficient in Na + (0.10%), supplemented with one of five levels of Na + (0.05, 0.10, 0.15, 0.20, or 0.25%) from NaHCO 3 resulting in diets with total levels of 0.10, 0.15, 0.20, 0.25, 0.30, and 0.35% of Na +. Diets (Table 1) were formulated to be essentially isonutritive, except for the test mineral, and to satisfy recommendations of the NRC (1994), with alterations by genetic line requirements. Feedstuff composition was based on Rostagno et al. (1987), NRC (1994), and results of the Animal Nutrition Lab. 5 The DEB was 3 Media Cybernetics, LP 8484, Georgia Avenue, Silver Spring, MD NAG Inc., Downers Grove, IL University Estadual de Maringá, Maringá, PR , Brazil. TABLE 2. Performance of growing chickens (21 to 42 d of age) fed diets with different levels of sodium or chloride Total sodium (%) or chloride (%) Variables Mean SEM Equation r 2 Experiment 1 (sodium) Body weight gain, g 1,563 a 1,590 a 1,580 a 1,492 ab 1,492 ab 1,433 b 1, Y ij = 1, X 2,943.16X Feed intake, g 2,972 2,982 2,972 2,915 2,911 2,895 2, Y ij = 3, X 0.83 Feed:gain ratio, g:g b b b ab ab a Y ij = X X Experiment 2 (chloride) Body weight gain, g 2,010 2,023 2,030 2,011 2,024 1,976 2, Y ij = 1, X 2,026.2X Feed intake, g 3,632 3,581 3,626 3,540 3,622 3,548 3, NS Feed:gain ratio, g:g Y ij = X X a,b Means within a row lacking a common superscript differ (P < 0.01). 1 NRC (1994) control treatments had 0.15% Na + or Cl in the respective studies.

4 292 MURAKAMI ET AL. TABLE 3. Arterial blood gas values of 35-d-old broiler chickens fed diets containing different levels of sodium or chloride Experiment 1 2 Experiment 2 Variable 1 Mean 3 SEM Mean 3 SEM Equation r 2 ph Y ij = X 0.98 PO 2, mm Hg NS PCO 2, mm Hg NS Bicarbonate, mm/l Y ij = X 0.84 TCO 2, mm/l Y ij = X 0.85 Base excess, meq/l Y ij = X 0.85 Oxygen saturation, % NS 1 PO 2 = Partial pressure of O 2 ;PCO 2 = partial pressure of CO 2 ;TCO 2 = CO 2 tension. 2 Regression equations were NS in Experiment 1. 2 Average of all treatments. calculated according to the procedure of Mongin (1980) (Table 1). The experimental design was completely randomized with six treatments, five replicates, and 50 birds per experimental unit. There were 30 litter pens total. Statistical analysis was performed with SAS software (1998), using ANOVA and mean separation with Tukey s multiple-range test. To estimate Na + requirement, quadratic and broken line models were used, choosing for conclusions the model that had the best statistical fit for each variable. Response curves were fitted using linear and quadratic regression procedures using PROC REG of the GLM procedure of SAS (1998). Experiment 2 This trial was conducted similarly to Experiment 1, differing in relationship to the mineral evaluated (Cl ). Average maximum and minimum temperatures were 25 ± 3.2 C and 17.5 ± 3.2 C. Initial chicken weight was 853 ± 19 g. Experiment 1 RESULTS AND DISCUSSION A quadratic effect (P 0.05) of Na + was observed on BWG and FCR, and a linear effect (P 0.01) was determined on FI (Table 2). Based on regression analyses, optimum Na + levels of 0.12 and 0.15%, respectively, were estimated for BWG and FCR. These values are similar to the recommendations of NRC (1994), Rostagno et al. (1996), and Murakami et al. (1997) and lower than those estimated by Barros et al. (1998). The very high levels of NaHCO 3 needed to achieve the highest Na + levels also contributed bicarbonate, which displaced some blood Cl and, conceivably, could have created a Cl deficiency or imbalance. A NaHCO 3 level of 0.60% gave normal results, but 0.79% or more was detrimental. No differences in arterial blood gas analyses (Table 3), were observed (P 0.05) in relation to Na + levels. Birds fed 0.20% Na + had a BE average of 1.80, the nearest to equilibrium (BE = 0) when compared with chickens in other treatments. Litter moisture increased linearly (P 0.01) as Na + dietary increased (Table 4), which caused a decrease in performance and an increase in breast blister incidence. Excessive Na + consumption causes increases in water intake, feces moisture, and Na + urinary excretion and a significant reduction in kidney glomerular filtration ratio (Vena et al., 1990). The TD incidence, evaluated by the method of Edwards and Veltmann (1983), was not affected by treatment (P 0.05) (Table 4). Values for growing cartilage (A1) and total area of epiphysis (A3) were normally distributed, but the hypertrophic zone of cartilage (A2) was not. All nonlinear models tested to describe these results did not significantly (P 0.05) explain the varia- TABLE 4. Litter moisture scores and tibial dyschondroplasia incidence of growing broiler chickens Total sodium (%) or chloride (%) Mean SEM Equation r 2 Experiment 1 Litter moisture score a 2.35 a 2.65 a 3.20 ab 3.40 b 3.35 b Y ij = X 0.85 Tibial dyschondroplasia NS Experiment 2 Litter moisture score NS Tibial dyschondroplasia NS a,b Means within a row lacking a common superscript differ (P < 0.001). 1 Litter moisture: 1 = litter is dry to 4 = litter is very damp. 2 Tibial dyschondroplasia score at 43 d of age by the method of Edwards and Veltmann (1983); 0 = normal cartilage; without or little irregularities; 1 = cartilage with some thickness or irregularity; 2 = thick cartilage; and 3 = big mass of cartilage not ossified.

5 SODIUM AND CHLORIDE REQUIREMENTS FOR BROILERS 293 FIGURE 1. Nonlinear model (gamma distribution and link function inverse power) describing areas of hypertrophic region of tibial growing cartilage of broiler chickens in relation to dietary chloride levels during growing phase at 42 d of age. The supplemental chloride (0.05 to 0.30%) was provided by sodium chloride (0 to 0.34%). tion of observed values. Murakami et al. (1997) concluded that Na + levels and Na + sources had no apparent effect on TD scores. Experiment 2 There was a quadratic, mound-shaped effect of Cl levels on BWG and FCR (P 0.05). The FI was not influenced (P 0.05) by Cl dietary levels. Based on regression equations (Table 2), we estimated that BWG was maximized when dietary Cl was 0.20% and FCR was minimized when Cl was 0.23%. The quadratic model was the best statistical fit to our data. However, the broken line model (not shown) demonstrated that at greater than 0.15% of dietary Cl, FCR was not further improved (P 0.05). This result confirmed NRC (1994) and Rostagno et al. (1996) recommendations, whereas these levels could still limit chicken performance and alter the acid-base balance. Means of blood gas parameters are presented in Table 3. A linear effect of Cl levels was observed on blood ph (P 0.05), HCO 3, TCO 2, and blood BE (P 0.01). These values diminished as dietary Cl increased, confirming observations of Adesina and Robbins (1987), Ruíz-López and Austic (1993), and Hooge (1998). Dietary Cl has an indirect acidogenic effect on bird metabolism. The BE was low with a tendency toward metabolic alkalosis (+1.47) in birds fed diets with 0.10% Cl ; however, slight metabolic acidosis ( 3.42) occurred in birds fed diets with 0.35% Cl. The apparent metabolic equilibrium (EB = 0) was at 0.19% of dietary Cl, and this value was similar to the level at which the best BWG occurred (Table 2). This finding confirms that the estimation of the Cl requirement is probably higher than 0.15% reported by other authors. Litter moisture scores were not affected (P 0.05) by dietary Cl levels (Table 4), which confirms that when dietary Cl is in adequate proportion with dietary Na + level, there are no significant increases in kidney excretion (Freeman, 1983). The TD incidence observed by the method described by Edwards and Veltmann (1983) did not show any significant differences (P 0.05) due to dietary Cl levels (Table 4); cartilage area determination verified that dietary Cl levels influenced TD incidence (P 0.001). A nonlinear model was plotted to describe this response (Figure 1). The model had gamma distribution and function link inverse power. At 0.30% dietary Cl, the hypertrophic cartilage area (A 2 ) increased significantly (P 0.001), which could have been associated with metabolic acidosis observed in birds fed this diet (Table 3). Similar results have been observed by Halley et al. (1987), Simons et al. (1987), and Ruíz-López et al. (1993). Chicken performance results as a function of DEB in the two experiments are presented in Table 5. In both experiments, a quadratic effect of DEB on bird performance was verified. Based on these equations, in Experiment 1, we estimated that the best BWG and FCR were obtained when birds were fed diets containing between 250 and 261 meq/kg, and in the Experiment 2, maximum BWG and minimum FCR were observed with diets containing between 249 and 257 meq/kg. In the Experiment 1, FI was depressed linearly by increasing DEB (P < 0.05); however, this influence was not observed in the Experiment 2. These results confirmed DEB recommendations for maximum growth by Saveur and Mongin (1978) and corroborated those of other authors (Patience, 1990; Hooge, 1995, 1998). Under the present conditions, dietary Na + and Cl requirements for growing broiler chickens, aiming for the best FCR, were 0.15 and 0.23%, respectively. The best DEB for growing broilers was between 249 and 261 meq/kg. These Na +,Cl, and DEB levels recommended did not affect acid-base balance status or TD incidence in broilers. The Cl levels higher than requirements estimated seemed to increase TD incidence. Litter moisture increased linearly as Na + dietary level increased, but Cl TABLE 5. Performance of growing broiler chickens in function of dietary electrolyte balance (Mongin, 1980) Experiment 1 Experiment 2 Equation r 2 DEB 1 Equation r 2 DEB 1 Body weight gain, g Y ij = X X Y ij = X X Feed intake, g Y ij = 3, X 0.75 NS Feed:gain ratio, g:g Y ij = X X Y ij = X X Dietary electrolyte balance = [Na + + K + ] Cl, meq/kg.

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