Requirement of calcium for commercial broilers and white leghorn layers at low dietary phosphorus levels

Animal Feed Science and Technology 106 (2003) 199–208

Requirement of calcium for commercial broilers and white leghorn layers at low dietary phosphorus levels S.V. Rama Rao∗ , A.K. Panda, M.V.L.N. Raju, G. Shyam Sunder, N.K. Praharaj Project Directorate on Poultry, Indian Council of Agricultural Research, Rajendranagar, Hyderabad 500030, India Received 28 August 2001; received in revised form 11 September 2002; accepted 11 September 2002

Abstract Two experiments were conducted to study the requirements of calcium (Ca) for commercial broilers (1–35 day of age) and White Leghorn (WL) layers (196–336 day of age) at a constant levels of phosphorus (P) recommended in the literature. In the first experiment, four levels of Ca (7, 8, 9 and 10 g kg−1 ) were tested in a maize–soya bean meal and de-oiled rice bran based diets at a constant level of non-phytin phosphorus (NPP) (3.5 g kg−1 ) with commercial broiler housed in stainless steel battery brooders. The experimental diets were fed ad libitum to 16 replicate groups of six broilers (three males and three female) in each replicate. Level of Ca in the diet significantly influenced the body weight gain (P ≤ 0.05), serum Ca level, tibia ash contents (P ≤ 0.01) and retention (P ≤ 0.05) of Ca and P. The level of Ca in diet did not affect the feed intake, feed per gain, leg abnormality score, net Ca retention and serum P content. The predicted requirements of Ca for maximum weight gain, serum Ca content and tibia ash content were 7.56, 9.72 and 9.83 g kg−1 diet, respectively. In the second experiment, six levels of Ca (32.5, 35, 37.5, 40, 42.5 and 45 g kg−1 ) were tested in a maize–soya bean meal–sunflower–de-oiled rice bran based diets containing a constant level of NPP (2.8 g kg−1 ) with layers. The experimental diets were fed ad libitum to six replicate groups (six layers in each replicate) housed in individual cages. Hen day egg production, feed intake, feed egg per mass, shell quality (shell thickness and shell weight), tibia ash content, tibia breaking strength and concentration of Ca and P in serum were not influenced by the level of Ca in the diet. The retention of Ca and P in the broiler experiment and the activity of serum alkaline phosphatase in the layer experiment were inversely related to the level of Ca in the diet. Based on the results, it can be concluded that the requirements of Ca for commercial broilers (1–35 day of age) and WL layers (196–336 day of age) are 7.56 and 32.5 g kg−1 diet, respectively. However, considering the trend seen with serum Ca concentration and tibia ash content in broilers and the activity of serum ∗ Corresponding author. Tel.: +91-40-4015651; fax: +91-40-4017002. E-mail address: [email protected] (S.V. Rama Rao).

0377-8401/03/$ – see front matter © 2003 Published by Elsevier Science B.V. PII: S 0 3 7 7 - 8 4 0 1 ( 0 2 ) 0 0 2 9 6 - 1

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alkaline phosphatase in layers, it may be desirable to provide 8 and 35 g Ca kg−1 in broiler and layer diets, respectively. © 2003 Published by Elsevier Science B.V. Keywords: Calcium; Requirement; Broiler; Layer

1. Introduction Current research on mineral nutrition is aimed at reducing the environmental pollution due to excretion of unutilized minerals, particularly phosphorus (P), from large scale poultry farming. The recent literature (Burnell et al., 1990; Scheideler et al., 1995; Gordon and Roland, 1997; Hossain and Bertechini, 1998; Rama Rao et al., 1999) suggest lower P requirements for commercial broilers (2.6–3.4 g kg−1 diet) and White Leghorn (WL) layers (2–2.5 g kg−1 diet). Since, the calcium (Ca) and P are co-existing in many biological functions, their dietary requirements are interdependent particularly in broilers (Hulan et al., 1985; Qian et al., 1997). Therefore, the requirements of Ca for poultry need to be established with changing P recommendations. In recent years, the productivity of broiler and layer chicken has increased considerably, perhaps due to changes in genetic potential of poultry and partly due to improved management practices. It is known that the genetic makeup of the bird influences the utilization of Ca (Shafey et al., 1990; Hurwitz et al., 1995) and thereby its requirement. Therefore, it is presumed that the requirement of Ca may not be the same as reported in earlier studies to meet the demand for highly productive birds at modified P recommendations. In the present study, it is proposed to study the Ca requirements of commercial broilers and WL layers at low dietary P levels as reported in the recent literature.

2. Materials and methods All the feed ingredients and compounded diets used in the present study were analysed for Ca (927.02) and crude protein (988.05) (AOAC, 1990). The phytin phosphorus (PP) content in feed ingredients was analysed (Haugh and Lantzsch, 1983). The non-phytin phosphorus (NPP) content in feed ingredients and compound diets was calculated as the difference between total phosphorus and PP. Two experiments were conducted to determine the Ca requirements for commercial broilers (1–35 day of age) and WL layers (196–336 day of age). Oyster shell grit was used as the source of Ca in both experiments. 2.1. Broiler experiment Three hundred and eighty four day-old Hubbard commercial broiler chicks were used in the first experiment. At day old, they were wing banded, weighed and randomly and equally distributed into 64 electrically heated raised wire floor stainless steel battery brooders (three males and three females per brooder). The chicks were vaccinated against Marek’s, Newcastle and fowl pox diseases as per the routine vaccination schedule. Four maize–soya

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Table 1 Composition of diets (g kg−1 ) varying in calcium levels fed to commercial broilers (1–35 day) Ingredient

Maize Soya bean meal De-oiled rice bran Oyster shell grit Di calcium phosphate DL methionine Choline chloride, 50% Common salt Vitamin premixa Trace mineral premixb Antibioticc Coccidiostatd Nutrient composition (g kg−1 ) Analyzed Crude protein Calcium Non-phytin phosphorus Calculated (g kg−1 ) Metabolizable energy (MJ kg−1 )

Calcium (g kg−1 ) 7

8

9

10

550.0 372.5 50.4 11.2 7.9 1.4 0.7 3.5 0.4 1.0 0.5 0.5

550.0 372.5 47.7 13.9 7.9 1.4 0.7 3.5 0.4 1.0 0.5 0.5

550.0 372.5 44.9 16.7 7.9 1.4 0.7 3.5 0.4 1.0 0.5 0.5

550.0 372.5 42.0 19.6 7.9 1.4 0.7 3.5 0.4 1.0 0.5 0.5

229.3 6.9 3.4

229.0 7.9 3.5

228.7 9.0 3.5

228.5 10.1 3.6

11.93

11.90

11.89

11.86

a

Vitamin premix provided (per kg diet): Vitamin A, 16,500 IU; Vitamin D, 3200 ICU; Vitamin E, 12 mg; K, 2 mg; thiamin, 1.2 mg; riboflavin, 10 mg; pyridoxine, 2.4 mg; cyanocobalamine, 12 mcg; niacin, 18 mg; pantothenic acid, 12 mg. b Supplies per kg of diet: MnSO , 40 g; ZnSO , 20 g; FeSO , 40 g; CuSO , 0.4 g; KI 0.03 g. 4 4 4 4 c Nitrofurazolidone 10% (w/w). d Monensine sodium 0.10% (w/w). Based on analyzed value of total and phytin phosphorus content of feed ingredients.

bean meal–de-oiled rice bran based diets were prepared to contain 7, 8, 9 and 10 g Ca kg−1 (Table 1). The desired levels of Ca in diets were obtained by adjusting the levels of oyster shell powder (<0.5 mm particle size) and de-oiled rice bran. The concentrations of other components (metabolizable energy, NPP, crude protein, lysine and methionine) were maintained constant across all the diets. Each diet was offered at random to ninety six chicks housed in 16 battery brooders (replicates) from 1 to 35 day of age. Feed and water were available ad libitum. Body weight and feed intake were recorded at weekly intervals throughout the experiment. At 36 day of age, the leg abnormality scores (Watson et al., 1970) of individual birds were recorded and one bird per replicate was picked up at random and slaughtered by cervical dislocation. Both the tibia were freed from soft tissue including diaphysis, then defatted by soaking in petroleum ether for 48 h. Dried bone samples were ashed at 600 ± 30 ◦ C for 12 h for estimation of bone ash. At 36 day, 3 ml blood was collected from one bird in each replicate. Sera samples were analyzed for Ca (927.02, AOAC, 1990) and P (Fiske and Subbarow, 1925) contents. A balance study of 3 day duration was conducted from 36 to 38 day of age to study the retention of Ca and P. Fifteen broilers were selected at random

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from each treatment and housed in five brooders at the rate of three birds per brooder (11 cm × 73 cm × 39 cm) to study the retention of Ca and P. Based on feed intake and excreta voided, the net retention of dry matter per unit intake of feed were calculated. The mineral retention was calculated as the difference between intake and output during the 3-day balance study. The retention was expressed as g per 100 g intake of the respective mineral. Further, the net retention of Ca was also expressed as the absolute quantity (g) of the mineral retained during the 3 day balance study. 2.2. Layer experiment Two hundred and sixteen commercial WL layers aged 28 weeks, with an average egg production of 87 eggs per 100 hens per day were randomly and equally distributed into six groups (36 layers in each group) with six replicates in each group (six birds per replicate). The birds were housed in individual California type cages (45.7 cm × 30 cm). Six experimental diets were formulated to contain 32.5, 35, 37.5, 40, 42.5 and 45 g Ca kg−1 diet utilizing common feed ingredients (Table 2). The NPP content was maintained constant at 2.8 g kg−1 diet. The levels of metabolizable energy, crude protein, lysine and methionine Table 2 Composition of diets (g kg−1 ) varying in calcium levels fed to White Leghorn layers (196–336 day) Ingredient

Yellow maize Soya bean meal Sunflower cake De-oiled rice bran Oster shell grit Dicalcium phosphate Common salt DL Methionine Vitamin premixa Trace mineral premixb Choline chloride, 50% Nutrient composition Analyzed (g kg−1 ) Crude Protein Calcium Non-phytin phosphorusc Calculated (g kg−1 ) Metabolizable energy (MJ kg−1 ) a

Calcium (g kg−1 ) 32.5

35.0

37.5

40.0

42.5

45.0

605.0 125.0 135 34.5

610.0 125.0 135 22.4

615.0 125.0 135 10.3

615.0 125.0 135 3.3

615.0 123.0 132 1.2

614.2 120 130 0.0

82.0 11.0 5.0 0.5 0.5 1.0 0.5

89.1 11.0 5.0 0.5 0.5 1.0 0.5

96.2 11.0 5.0 0.5 0.5 1.0 0.5

103.2 11.0 5.0 0.5 0.5 1.0 0.5

110.3 11.0 5.0 0.5 0.5 1.0 0.5

117.3 11.0 5.0 0.5 0.5 1.0 0.5

160.2 32.6 2.8

159.2 35.4 2.8

158.5 37.6 2.8

157.8 39.8 2.8

156.2 42.4 2.8

155.8 45.2 2.8

10.90

10.88

10.84

10.80

10.73

10.62

Supplies per kg diet: Vitamin A, 16,500 IU; Vitamin D3 , 3200 ICU; Vitamin E, 12 mg; Vitamin K, 2 mg; Vitamin B1 , 1.2 mg; Vitamin B2 10 mg; Vitamin B6 , 2.4 mg; Vitamin B12 , 12 ␮g ; niacin, 18 mg; pantothenic acid, 12 mg. b Mn, 50 mg; Zn, 112.5 mg; Fe, 60 mg; Cu, 10 mg; I, 1.2 mg. c Based on analyzed value of total and phytin phosphorus content of feed ingredients.

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were kept constant in all diets. The levels of ground oyster shell (25:75, powder and grit 1–2 mm, respectively), de-oiled rice bran and maize were adjusted to arrive at the desired levels of nutrients in the diets. Each diet was allotted at random to six replicates and offered ad libitum from 196 to 336 day of age. Light was provided 16 h daily using incandescent bulbs. The maximum and minimum temperature during the experimental period ranged between 30–34 and 18–23 ◦ C, respectively. Hen day egg production, feed intake and individual body weight gain during the experimental period were recorded. All the eggs laid during the last three consecutive days of every 28 days period were weighed. From this sample six eggs were randomly chosen daily from each treatment to determine the shell quality (shell thickness and shell weight). The mean of the twelve eggs was considered as a replicate (six replicates in each treatment) to determine the shell weight and shell thickness. The shell thickness was measured at three different locations (both ends and at the middle) with a micrometer gauge (Mitutoyo, Code no. 7301, Japan). At the end of every 28 days period (224, 252, 280, 308 and 336 day of age), one bird per replicate was chosen randomly (five values per a replicate) and 5 ml of blood sample was collected from brachial vein at 14.30–15.30 h to estimate the serum Ca (927.02, AOAC, 1990), P (Fiske and Subbarow, 1925) and activity of alkaline phosphatase using diagnostic kit (P. no. 72011, Qualigens Diagnostics, India). At 337 day of age, one bird from each replicate (six birds per a dietary Ca level) were selected at random and slaughtered by cervical dislocation. The tibia was collected from each bird to estimate the tibia breaking strength with Universal Testing Machine (EZ Test, Shimadzu, Japan) and bone ash content as in the broiler experiment. The data were subjected to polynomial analysis (Snedecor and Cochran, 1980) using General Linear Model (SAS, 1995). The response in the criteria considered were fitted by polynomial equation in the form Y = a + bx + cx2 + dx3 . When the R2 of a criterion was significant, the requirement of Ca for maximum response of the trait was calculated. 3. Results and discussion 3.1. Broiler experiment The performance of commercial broilers from 1 to 35 day of age on diets with different levels of Ca is presented in Table 3. The R2 for body weight gain, serum Ca level, tibia ash content and retention of Ca and P were significant. The feed intake, feed per gain, leg score, serum P content and net retention of Ca were not significantly (P > 0.05) influenced by the level of Ca in diet. The predicted Ca requirement for maximum body weight gain was 7.56 g kg−1 diet. Further increase in dietary Ca level showed a declining (P ≤ 0.01) response for growth. Therefore, the results indicate that the commercial broilers need 7.56 g Ca kg−1 for maximum growth when the diet contained 3.5 g NPP kg−1 . These findings are in line with the earlier reports (Sohail and Roland, 1999; Boling-Frankenbach et al., 2001), who suggested the Ca requirement for broilers about 7.5 g kg−1 diet. The growth depression observed at higher levels of Ca in the present study might be due to the wider Ca and NPP ratio (2.85:1). A Ca and NPP ratio beyond 2:1 is known to reduce the bio-availability of Ca and P by forming an insoluble calcium phosphate complex in the

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Table 3 Effect of different dietary levels of calcium on various production traits and predicted requirement of calcium for different parameters in commercial broilers (1–35 day of age) Attribute

Body weight gain (g) Feed intake (g) Feed per gain Leg score Serum Ca (mg dl−1 ) Serum P (mg dl−1 ) Tibia ash (g kg−1 ) Ca retention (g per 100 g) Net Ca retention (g per 3 days) P retention (g per 100 g)

Calcium (g kg−1 )

S.E.M. (±)

N

R2

Ca required predicted (g/kg)

0.277∗ 0.047 0.098 0.025 0.256∗ 0.092 0.737∗ 0.953∗∗

7.56 – – – 9.72 – 9.83

7.0

8.0

9.0

10.0

1263 2558 2.02 2.3 9.21 5.86 48.2 583

1264 2378 1.86 2.3 10.56 5.80 52.1 553

1213 2430 2.01 2.5 10.23 5.75 53.5 517

1088 2290 2.11 2.5 10.49 5.84 54.4 451

17.23 42.00 0.03 0.06 0.12 0.5 0.64 11.5

16 16 16 16 5 5 5 5

1.67

1.64

1.70

1.58

0.16

5

0.089

–

1.20

5

0.987∗∗

a

38.2

34.3

31.3

24.6

a

a

Retention of Ca and P increased linearly with decrease in the levels of Ca in the diet. Significant at P < 0.05. ∗∗ Significant at P < 0.01. ∗

chicken gut which adsorbs certain essential trace minerals like Mn, Zn, Cu, etc. (Underwood, 1981). The significant (P < 0.01) reduction in the retention of Ca and P with increases in the dietary Ca from 7 to 10 g kg−1 (Table 3) also indicates the formation of an insoluble complex (calcium phosphate) in the gut at higher dietary Ca level. Contrary to the present findings, Hulan et al. (1985) and Mitchell and Edwards (1996) reported higher Ca levels (13 and 9.8 g kg−1 diet, respectively) as the requirement for commercial broilers. The higher Ca requirements suggested by these authors perhaps might be due to higher P levels used in their studies (6.8 and 4.5 g kg−1 diet, respectively). The dietary level of NPP is known to influence the Ca requirements in broilers. Therefore, it appears that dietary Ca levels can be reduced to 7.56 g kg−1 by maintaining the Ca and NPP ratio at around 2:1 in the diet, which is about 24.4% less than that recommended by NRC (1994) (10 g kg−1 diet) for broiler starter diets. The serum Ca level increased significantly (P ≤ 0.05) with increases in the dietary Ca level, corroborating with the findings of Hurwitz et al. (1995). The predicted Ca requirement for maximum serum Ca level was 9.72 g kg−1 diet. The level of Ca in the diet did not influence the concentration of P in the serum. The tibia ash content increased significantly (P ≤ 0.05) with increasing levels of Ca in the diet (Table 3). The predicted requirement of Ca for maximum bone mineralization was 9.83 g kg−1 diet, suggesting that the requirement of Ca for bone formation during the juvenile stage was higher than the levels required for optimum body weight. Similarly, earlier reports (Rama Rao et al., 1999) suggested higher requirement of minerals for skeletal development compared to optimum growth. Though the data show that broilers fed 7 and 8 g Ca gained similar weights, the statistical analysis indicates the maximum weight gain (1269 g) at 7.56 g Ca/kg diet compared to those fed 7

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and 8 g Ca/kg diet. Although the bone ash content was maximum at 9.83 g Ca kg−1 diet, the leg abnormality was not affected due to variation in dietary Ca level. The trends of change in feed per gain, serum Ca concentration and tibia ash content suggest higher Ca levels (8 g kg−1 ) as the requirement in practical broiler diet. Retention of Ca and P decreased significantly (P ≤ 0.01) with increasing levels of Ca in the diet (Table 3). The predicted equations indicated significant inverse relation between the retention of these minerals and dietary Ca levels. The prediction equation indicates the retention of Ca and P to be maximum at the lowest possible level of dietary Ca (i.e. <0.01 g kg−1 ). However, biologically it may not be possible to incorporate Ca at very low levels to achieve maximum retention of these minerals in commercial broilers. The significant decrease in the retentions of Ca and P at higher dietary Ca levels (higher Ca and NPP ratio) also suggests the possibility of formation of calcium phosphate in the gut with consequent reduction in the absorption of these minerals from the intestine. 3.2. Layer experiment The production performance of WL layers (196–336 day of age) fed with different dietary Ca concentration is summarized in Table 4. The hen day egg production varied between 93.5 and 94.2, 100 per birds and did not differ significantly (P ≥ 0.05) among the layers fed with different levels of Ca. Similarly, Ca levels greater than 32.5 g kg−1 diet had no affect on egg weight, feed egg per mass eggshell quality (shell weight and shell thickness), tibia ash content, tibia breaking strength and serum Ca and P levels. Contrary to the present findings, El-Boushy and Papadopoulos (1979) reported a significant increase in tibia ash content and tibia breaking strength with increase in dietary Ca levels (37 versus 50 g kg−1 ). The significant increase in bone parameters in their study might be due to large variations in the Ca levels tested. Since the lowest Ca level used in the present experiment (32.5 g kg−1 diet) could meet requirement, higher levels did not elicit any further response in bone mineralization parameters (tibia ash and tibia breaking strength). These results indicate that WL layers do not require more than 32.5 g Ca kg−1 diet during the peak phase of production. Contrary to this finding, few reports indicate higher requirements (34–45 g kg−1 diet) of Ca for layers. Although there is variation in current Ca recommendations when expressed as the concentration of the diet in previous studies (Keshavarz and Nakajima, 1993; Leeson et al., 1993), the absolute intake of Ca (3.4–3.72 g per bird per day) is similar to that of the present study (3.51 g per bird per day). The lower requirement of Ca as suggested by Roush et al. (1986) might be due to relatively lower egg production potential (79 eggs 100 per layers) of birds used in their studies. Since the genetic variation has been reported in utilization of Ca in poultry (Edwards, 1983), it is presumed that wide genetic diversity in birds used in the previous studies might be responsible for variations observed in defining the optimum Ca requirement for layers. The variation also might be due to differences in age of the bird (Keshavaraz and Nakajima, 1993), environmental temperature (Kamar et al., 1987) and particle size (Rao and Roland, 1989) of Ca supplements. The variation in level of Ca in diet did not influence (P > 0.05) the serum Ca and P levels. However, the level of Ca in the diet significantly (P ≤ 0.01) influenced the activity of serum alkaline phosphatase (Table 4) which decreased with increased Ca level in the diet. The maximum enzyme activity was observed at the lowest level of Ca in the diet as low

206

Attribute

Hen day egg production (per 100 layers) Egg weight (g) Feed intake per day (g) Feed egg per mass Tibia ash (g kg−1 ) Tibia breaking strength (N) Egg shell weight (g kg−1 ) Egg shell thickness (mm) Serum Ca (mg/dl) Serum inorganic P (mg/dl) Serum alkaline phosphatase (KA units) a b

Calcium (g kg−1 ) 32.5

35.0

37.5

40.0

42.5

45.0

94.1 54.6 107.9 2.11 519 691 98.1 0.385 24.2 6.83 55.1

93.9 55.3 106.0 2.11 532 709 99.3 0.379 25.2 6.93 31.4

93.5 54.7 106.3 2.11 535 721 98.2 0.386 24.5 6.99 25.7

93.9 54.9 106.2 2.11 535 722 98.9 0.391 25.0 6.87 19.2

93.5 55.2 106.7 2.12 536 723 98.4 0.386 25.1 7.01 31.0

94.2 54.8 107.0 2.12 533 719 98.3 0.389 25.1 7.07 25.3

Significant at P < 0.01. The activity of serum alkaline phosphatase decrease with increase in the levels of Ca in the diet.

S.E.M. (±)

N

R2

0.26 0.10 0.37 0.01 6.1 13.0 0.4 0.004 0.37 0.13 1.09

6 6 6 6 6 6 6 6 6 6 6

0.130 0.073 0.156 0.011 0.152 0.122 0.047 0.032 0.070 0.025 0.835a

Ca required predicted (g/kg) – – – – – – – – – – b

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Table 4 Effect of different dietary levels of calcium on various production traits on WL layers (196–336 day)

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dietary Ca is known to increase bone resorption by releasing more alkaline phosphatase. Concomitant to the findings of the experiment, Reichmann and Connor (1977) also reported lower activity of serum alkaline phosphatase at higher dietary Ca levels. Since the egg production, feed intake, egg weight and shell quality were similar at different levels of Ca tested (32.5–45 g kg−1 diet), the lowest level of Ca tested, i.e. 32.5 g kg−1 diet, may be considered as the requirement for layers during 196–336 day of age. Based on the data, it is concluded that commercial broilers (1–35 day of age) required 7.56 g Ca kg−1 diet when the diet contained 3.5 g NPP kg−1 for optimum growth, feed per gain and leg score, while the WL layers (196–336 day of age) require 32.5 g Ca kg−1 diet for optimum egg production and shell quality. However, considering the trend seen with serum Ca concentration and tibia ash content in broilers and the duration of experiment conducted and the activity of serum alkaline phosphatase in layers, it may be desirable to provide 8 and 35 g Ca kg−1 in broiler and layer diets, respectively.

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