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International Journal of Morphology

versión On-line ISSN 0717-9502

Int. J. Morphol. v.21 n.2 Temuco  2003

http://dx.doi.org/10.4067/S0717-95022003000200001 

Int. J. Morphol., 21(2):101-106, 2003.

BONE DEVELOPMENT OF BROILER CHICKENS FED DIETS WITH DIFFERENT 
AMINO ACID AND CALCIUM LEVELS DURING THE STARTER PHASE

DESARROLLO ÓSEO DE POLLOS DE CORTE QUE CONSUMEN DIFERENTES NIVELESDE AMINOÁCIDOS Y DE CALCIO EN LA FASE INICIAL DE CRÍA

*Cristiane Soares da Silva Araújo; *Silvana Martinez Baraldi Artoni; *Lúcio Francelino Araújo;
**Mário Jeferson Quirino Lousada; *Otto Mack Junqueira & *Anita Isabel Roque Rodriguez


SUMMARY: Two experiments were conducted in order to evaluate the effects of different dietary amino acid and calcium profiles on the bone characteristics of two chicken strains (Avian Farms and Cobb) during the starter phase. At the end of each experiment, 2 chickens of each replicate were sacrificed and their tibiae were collected for analysis of following bone characteristics: bone measurements (weight, length and thickness of the compact and spongy layers) and bone density (bone densitometry). A total of 540 birds were used in each experiment, divided into fully randomized blocks with a 3 x 2 factorial schemes, i. e., 3 amino acid profiles (methionine, lysine and threonine ­ 100%, 125% and 150% of NRC recommendations, 1994) and 2 calcium levels (75% and 100% of NRC recommendations, 1994), with 30 birds per replicate. Birds of the Cobb strains fed 75% of the recommended calcium allowance showed lower bone density. Calcium level significantly affected the weight and thickness of the spongy layer of the tibia. In birds of the Avian strain, bone density and measurements were not affected by any of the experimental treatments.

KEY WORDS: 1. Amino acid; 2. Bone density; 3. Broiler chickens; 4. Calcium. 


INTRODUCTION

The search for new ways to improve the quality of products of animal origin is a definite trend in food production. Within this context, the genetic potential of broiler chickens has been rapidly improved, with an increased demand for post-processed chicken products. On this basis, there is currently great interest in the composition and quality of chicken carcasses, especially in terms of meat yield and composition. A necessary and desirable characteristic is a low amount of fat, since its total content reaches about 2,5% (Holsheimer & Veerkamp, 1992). Fraps (1943) was one of the first investigators to demonstrate that carcass composition can be manipulated through the energy and protein levels of the diet.

Nutrition is a considerable factor accounting for successful aviculture. Commercial sources of synthetic methionine, lysine and threonine are available on the market and have been used in rations to satisfy chicken requirements in terms of the first limiting amino acids. When the protein level or even the lysine level of the diet is increased in isoenergetic diets, protein retention is increased and the amount of carcass fat is reduced (Bedford & Summers, 1985).

However, in parallel to these advances in performance, the animals have experienced leg problems due to this intense development. Despite countless efforts on the part of the aviculture industry, this continues to be a serious situation, since chickens are unable to support their own weight. Thus, it is important to consider the amino acid levels used in the diet for broiler chickens. Unfortunately, with the preoccupation of reaching better zootechnic performance, little or no attention has been paid to the effects of excessive amino acid amounts on bone mineralization in broiler chickens.

In a study on broiler chickens, Skinner et al. (1991) observed that when the total amino acid levels were increased by 20% above the requirements of the birds, a significant reduction in tibia mineralization occurred among birds consuming diets containing 0,5% and 1,0% calcium. Thus, there is now a new concern about the use of synthetic amino acids since excessive level can cause an increased incidence of leg abnormalities, which in turn cause a reduction in growth and high morbidity, representing one of the great problems of aviculture worldwide because of the economic losses observed.

In view of the above considerations, the objective of the present study was to evaluated the effects of different amino acid (methionine, lysine and threonine) and calcium levels on the bone characteristics of two commercial strains of broiler chickens during the starter phase.

MATERIAL AND METHOD

Two experiments were carried out at the experimental aviary of the Faculty of Agrarian and Veterinary Sciences, UNESP, Jaboticabal ­ Brazil, in order to evaluate the bone characteristics of two strains of broiler chickens (Avian Farms and Cobb) during the starter phase from 1 to 21 days of age. A total of 540 birds were used in each experiment, divided into fully randomized blocks with a 3 x 2 factorial schemes, i. e., 3 amino acid profiles (methionine, lysine and threonine ­ 100%, 125% and 150% of NRC recommendations (1994) and 2 calcium levels (75% and 100% of NRC recom-mendations, 1994) with 30 birds per replicate. The experimental rations consisted of corn, soy bran, bicalcium phosphate, limestone, salt, synthetic amino acid and calcium levels established by NRC (1994). The dietary variables were the amino acid profiles and calcium levels (Table I).

Table I. Percent composition of the experimental diets for broiler chickens submitted to different dietary amino acid and calcium levels during the starter phase.


Ingredients Experimental rations
  100%Ca
100% AA
100%Ca
125% AA
100%Ca
150% AA
  75% Ca
100% AA
75% Ca
125% AA
75% Ca
150% AA

Corn 49. 84 49. 84 49. 84   50.50 50.50 50.50
Soy oil 39.00 39.00 39.00   39.00 39.00 39.00
Soy bran 05.70 05.70 05.70   05.70 05.70 05.70
Bicalcium phosphate 01.74 01.74 01.74   01.74 01.74 01.74
Limestone 01.13 01.13 01.13   00.47 00.47 00.47
Salt 00.39 00.39 00.39   00.39 00.39 00.39
DL-Methionine 00.20 00.45 00.70   00.20 00.45 00.70
L-Lysine 00.00 00.25 00.50   00.00 00.25 00.50
L-Threonine 00.00 00.25 00.50   00.00 00.25 00.50
Vit + mineral suppl. 00.50 00.50 00.50   00.50 00.50 00.50
Inert 01.50 00.75 00.00   01.50 00.75 00.00
Total 100.000 100.000 100.000   100.000 100.000 100.000
  Calculated Analysis
EM (kcal/kg) 3.100 3.100 3.100   3.100 3.100 3.100
PB (%) 22.000 22.000 22.000   22.000 22.000 22.000
Ca (%) 1.00 1.00 1.00   0.75 0.75 0.75
Pdisp(%) 0.44 0.44 0.44   0.44 0.44 0.44
Met (%) 0.54 0.67 0.81   0.54 0.67 0.81
Met+Cist(%) 0.90 1.12 1.35   0.90 1.12 1.35
Lis(%) 1.10 1.37 1.65   1.10 1.37 1.65
Tre(%) 0.87 1.08 1.30   0.87 1.08 1.30

Guaranteed levels of vitamin and mineral supplements per kg product: vit. A: 300.000 UI; vit. D3: 100.000 UI; vit. E : 4.000 mg; vit. K: 98 mg; vit. B1: 400 mg; vit. B2:1.320 mg; vit. B12: 4.000 mcg; pantohtenate: 2.000 mg; niacin: 20.000 mg; folic acid: 100 mg; growth promoter: 10.000 mg; choline: 50.000 mg; copper: 15.000 mg; iodine: 250 mg; selenium: 50 mg; manganese: 24.000 mg; zinc: 20.000 mg; iron: 10.000 mg; coccicide: 25.000 mg; antioxidant: 125 mg and vehicle q. s. p.: 1.000 g.

At the end of each experiment (21 days), two birds from each replicate were sacrificed for leg removal and evaluation of the bone characteristics of the tibiae, i. e., bone measurements (weight, length and thickness of the compact and spongy layers) and bone density (bone densitometry). The legs were stripped of flesh and the tibiae were obtained for evaluation of bone measurements. A precision scale was used toweigh the tibiae and the measurements of bone length and of compact and spongy layer thickness were made with the aid of a pachymeter, after performing a median sagittal cut in the tibia.

Bone density was measured by densitometry using radiographic images of the middle third of the tibia in the medullary canal. As a densitometry reference an aluminum scale (6063 alloy, ABNT) and 12 steps (a 0,5 mm thickness for the first step with successive 0,5 mm steps up to the tenth, 6.0 mm thickness for the eleventh and 8.0 mm thickness for the twelfth, each step had an area of 5 x 25 mm2) was radiographed together with the bone pieces. A standard x-ray machine was calibrated for 55 kVp and 5 mAs, with a 1 m distance between the focus and the film. The X-ray films used were of The BRAF brand, not necessarily of the same lot. Development and fixation were carried out using a standard automatic processing apparatus, according to routine clinical radiologic procedures.

For the densitometry readings we first used an HP Scanjet 4C scanner equipped with an HP Scanjet 4C transparency adaptor for digitalization of the radiographic images. After image digitalization using the HP DeskScan program, the images were stored in a microcomputer. A computation program was used to recapture the images and a region of each was selected for analysis with the aid of the mouse. After the region was determined the program read the data and provided the optic density values expressed as grey levels (up to 256). This program was constructed in such a way as to permit selection of the area to be used for densitometry readings. Once the area to be used was defined, the program read it pixel by pixel, providing the optic density value as the mean of these readings. The optic density values obtained in all radiographs of the bone images and of an aluminum scale were stored in the microcomputer and processed for the pertinent analyses. The densitometric values obtained with the HP Scanjet 4C scanner were converted to thickness values, with the computer using the methodology described by Louzada (1994).

Data were analyzed statistically with the ESTAT, program development by the Department of Exact Sciences of UNESP/FCAVJ. The Tukey test was used for comparison of the means.

RESULTS

Bone density: Bone density data at 21 days of age for birds of the Cobb strain are presented in Table II. No significant interaction was observed between the 2 factors and the amino acid levels did not affect this characteristic. However, birds receiving a diet containing 75% calcium presented a lower bone density density (P<0,05). Although the lowest calcium level in the diet led to lower bone density as mentioned earlier the performance of this strains was nor significantly affected by this factor, probably because this calcium level was not sufficiently low to impair the productive traits.

Table II. Bone density (expressed as mm de aluminium equivalents) of broilers chickens of the Cobb strais fed different amino acid and calcium levels at 21 days of age.


  Amino acids (%)
Calcium (%) 100 125 150 Mean
00075 1,92 2,09 1,97 1,99 b
000100 2,30 2,45 2,27 2,34 a
Mean 002,11 A 002,27 A 002,12 A  
  (CV= 7,53)      

Means followed by the same letter along the same line did not differ significantly from one another by the Tukey test(P > 0,05).

No interaction between the factors studied occurred with respect to the bone density of the Avian Farms strain (Table III). Amino acid levels had no effect on densitometry. In addition, in contrast to what was observed for Cobb birds during the same period. Calcium levels did not influence this parameter.

Table III. Bone density (expressed as mm de aluminium equivalents) of broilers chickens of the Avian Farms strais fed different amino acid and calcium levels at 21 days of age.


  Amino acids (%)
Calcium (%) 100 125 150 Mean
00000075 2,15 2,42 1,99 2,18 a
000000100 2,30 2,19 2,04 2,19 a
Mean 2,22 A 2,31 A 2,02 A  
  (CV= 13,63)      

Means followed by the same letter along the same line did not differ significantly from one another by the Tukey test (P > 0,05).

Bone measurements: The bone measurements of the tibia of Cobb chickens during the starter phase are listed in Table IV. No interaction between amino acid and calcium levels was observed for any of the characteristics studied. On the other hand, length measurements tended to decreases, even though in a non significant manner, with increasing amino acid levels. The opposite occurred with mean thickness of the compact layer. As also observed for density, some bone measurement presented significantly worse means when the birds consumed diets containing 75% calcium. This was the case for tibia weight and spongy layer thickness which were significantly affected (P<0,05) by calcium levels, with better results being obtained when calcium levels were 100% of the recommendations.

The bone measurements of Avian Farms chickens (Table IV) did not show interaction of amino acid and calcium levels. The length, weight and thickness of the compact and spongy layers of the tibia were not affected by any of the factors studied.

Table IV. Length, weigth and thickness of the compact and spongy layers of the tibia of broiler chickens of the Cobb strain fed different amino acid and calcium levels at 21 days of age.


Parameter   Amino acids (%)
  Calcium (%) 100 125 150 Mean
Tibia 75 7,12 6,96 6,97 7,02 a
Length(cm) 100 6,98 6,98 7,00 6,98 a
  Mean 7,05 A 6,97 A 6,98 A  
  (CV= 3,15)        
Tibia 75 5,43 5,64 5,63 5,57 b
Weight (g) 100 6,17 6,28 6,71 6,38 a
  Mean 5,80 A 5,96 A 6,17 A  
  (CV=11,68)        
Compact 75 1,67 1,79 1,79 1,75 a
Layer 100 1,87 1,87 2,04 1,93 a
Thickness (mm) Mean 1,77 A 1,83 A 1,91 A  
  (CV=13,21)        
Spongy 75 9,58 10,50 10,58 10,22 b
Layer 100 10,50 10,50 A 10,75 10,92 A
Thickness (mm) Mean 10,04 A 10,50 A 10,17 a  
  (CV=5,52)        

Means followed by the same letter along the same line did not differ significantly from one another by the Tukey test (P > 0,05).

Table V. Length, weigth and thickness of the compact and spongy layers of the tibia of broiler chickens of the Avian Farms strain fed different amino acid and calcium levels at 21 days of age.


Parameter   Amino acids (%)
  Calcium (%) 100 125 150 Mean
Tibia 75 6,80 6,90 7,00 6,90 a
Length(cm) 100 6,80 6,80 7,00 6,86 a
  Mean 6,80 A 6,85 A 7,00 A  
  (CV= 4,06)        
Tibia 75 5,76 6,25 6,15 6,05 a
Weight (g) 100 5,78 5,53 6,44 5,92 a
  Mean 5,77 A 5,89 A 6,29 A  
  (CV=13,28)        
Compact 75 1,74 1,83 1,50 1,69 a
Layer 100 1,87 1,92 1,54 1,78 a
Thickness (mm) Mean 1,80 A 1,87 A 1,52 A  
  (CV=12,54)        
Spongy 75 10,33 10,46 10,83 10,54 a
Layer 100 10,33 10,71 11,00 10,68 a
Thickness (mm) Mean 10,33 A 10,58 A 10,92 A  
  (CV=6,50)        

Means followed by the same letter along the same line did not differ significantly from one another by the Tukey test (P > 0,05).

DISCUSSION

Few studies related to densitometric bone analysis in aviculture are available. Another aggravating factor in the scarcity of literature relating amino acid and calcium levels to bone density.

In a study of bone development of broiler chickens fed different amino acid and calcium levels, with respective recommendations of 80, 100 and 120% and 50, 100 and 150% of NRC guidelines (1984), Sekine et al. (1994) noted an interaction between the factors evaluated and bone density. Diets containing 150% of recommended calcium levels did not promote a greater resistance of the tibia to fracture compared to rations containing 120% amino acids. In addition, calcium levels affected the longitudinal growth of the tibia and promoted bone calcification. On the other hand, amino acid levels acted on transverse growth and facilitated the formation of bone matrix as well as calcification.

When González et al. (1993) evaluated the effects of amino acid and calcium levels on bone mineralization, they observed that neither factor affected bone development. These investigators stated that birds with a high growth rate are more susceptible to diets containing low calcium levels for adequate mineralization. This was the case for Cobb birds, which presented lower bone density when fed diets with lower calcium levels, probably due to the high initial growth rate compared to Avian Farms bird.

Some investigators have reported that the excess of amino acids in diets for broiler chickens can affect bone development. Also, El-Marghi et al. (1965) noted severe osteoporosis in rats consuming diets with a high protein contend but a low calcium contend. However, Bell et al. (1975) reported that diets with high protein levels may not affect bone resorption in rats when adequate calcium and phosphorus amounts are ingested. Later González et al. noted a reduction in tibia weight with increasing sulfur amino acids levels (0.93, 1.23 and 1.53%). The cited authors stated that this was an effect apparently associated with the reduction in growth in growth rate and nor with the increase in the catabolism of these nutrients.

With respect to calcium levels, Nelson et al. (1990), in a study of the relation between mineral and the incidence of leg abnormalities, reported in a fist experiment that the reduction of leg problems was not significantly affected by increased calcium levels (0.6, 0.9 and 1.20%). However, in a second experiment an increase in calcium from 0.9 to 1.20% caused a significant reduction of these abnormalities. Elliot et al. (1992, 1995) obtained similar results showing that the elevation of calcium from 0.65 to 0.90% promoted a decrease of tibia dyschondroplasia.

Studying different amino acid and calcium levels, Skinner et al. observed that tibia weight was affected by calcium levels and, in contrast to what occurred in the present study, there was a significant interaction between amino acid and calcium levels for this measurement. When the birds consumed 1.0% calcium in the diet, tibia weight increased with decreasing amino acid levels, however, when the diet contained 0.50% calcium no increase in tibia weight occurred with elevation in amino acid levels.

The low calcium level for the Cobb strains reduced bone density, tibia weight and spongy layer thickness, demonstrating that this strains was more sensitive to a reduction in dietary calcium. It would be interesting to conduct additional studies to evaluate the influence of dietary amino acid excess on performance and bone development.

The technique of bone density evaluation by radiographic optic density should be used more frequently since it can be an alternative method for the analysis of bone development in broiler chickens. 


RESUMEN: Se realizaron dos experimentos con el objetivo de analizar los efectos de los diferentes niveles de aminoácidos y de calcio sobre las características óseas de dos líneas de pollos de corte (Avian Farms y Cobb) en la fase inicial de cría. Al final de cada experimento, fueron sacrificadas 2 aves de cada repetición, para colectar las tibias y analizar sus características: peso, largo, grosor de las capas óseas compacta y esponjosa) y la densidad ósea (densitometríaósea). En cada experimento, fueron utilizadas 540 aves, distribuidas al azar, en esquema factorial 3x2, que significa, 3 perfiles de aminoácidos (metionina, lisina y treonina: 100%, 125% y 150% NRC, 1994) y 2 niveles de calcio (75% y 100% NRC, 1994), con 30 aves por repetición. Las aves de la línea Cobb alimentadas con 75% de lo recomendado de calcio presentaron menor densidad ósea. El nivel de calcio, influyó significativamente en el peso y grosor de la capa esponjosa de la tibia. En la línea Avian Farms, los tratamientos aplicados no afectaron la densidad ni las medidas óseas.

PALABRAS CLAVE: 1. Aminoácidos; 2. Calcio; 3. Densidad ósea; 4. Pollos de corte. 


REFERENCES

Bedford, M. R. & Summers, J. D. Influence of the ratio of essential and non essential amino acids on performance and carcass composition of the broiler chick. Br. Poult. Sci., 26:483-91, 1985.         [ Links ]

Bell, R. R.; Engelmann, D. T.; Sie, T. & Draper, H. H. Effect of a high protein intake on calcium metabolism in the rat. J. Nutr., 105:475-83, 1975.         [ Links ]

Elliot, M. A. & Edwards, H. M. Studies to determine wether an interaction exists among boron, calcium and cholecalciferol on the skeletal development of broiler chickens. Poult. Sci., 71:667-90, 1992.         [ Links ]

Elliot, M. A.; Roberson, K. D.; Rowland, G.N. & Edwards, H. M. Effects of calcium and 1,25 dihydroxy-cholecalciferol on the development of tibial dyscondroplasia in broiler during the starter and grower periods. Poult. Sci., 74:1495-505, 1995.         [ Links ]

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Louzada, M. J. Q. Otimização da técnica de densitometria óptica em imagens radiográficas de peças ósseas. Estudo "In Vitro". Tese de Doutorado, UNICAMP, Campinas, SP, Brasil. 191p. 1994.         [ Links ]

Nelson, T. S.; Kirby, L. K & Johnson, Z. B. Effect of minerals on the incidence of leg abnormalities in growing broiler chickens. Nut. Res., 10:525-33, 1990.

NRC - National Research Council. Nutrient requirements of Poultry. Washington: Natl. Acad. Press, 8th revised ed., 1984.         [ Links ]

NRC - National Research Council. Nutrient requirements of Poultry. Washington: Natl. Acad. Press, 9th revised ed., 1994.         [ Links ]

Sekine, T.; Watanabe, E & Ishibashi, T. Influence of dietary amino acids and calcium-available phosphorus on bone development of female broiler chicks. Anim. Sci. Techn. 65:999-1007, 1994.         [ Links ]

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Correspondence to:
Prof. Dra. Silvana Martinez Baraldi Artoni
Departamento de Morfologia e Fisiologia Animal
Universidade Estadual Paulista - UNESP
Via de Acesso Prof. Paulo Donato Castellani, Km 5 s/n
CEP 14870-000
Jaboticabal - SP
BRASIL
Email:smbart@fcav.unesp.br

Received : 31 december 2002
Accepted : 27 january 2003 


* FCAV/UNESP ­ Jaboticabal/SP., Brazil.
** Dental School, UNESP ­ Araçatuba/SP., Brazil.
Research supported by Fundation of Ampare of Research of São Paulo State (FAPESP), Brazil (98/15958-3).

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