PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION. Densitometric and biochemical values of broiler tibias at different ages

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1 PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Densitometric and biochemical values of broiler tibias at different ages F. R. Barreiro,* 1 A. L. Sagula,* O. M. Junqueira, G. T. Pereira, and S. M. Baraldi-Artoni * * Department of Animal Morphology and Physiology, Department of Animal Sciences, and Department of Statistical Analysis, Universidade Estadual Paulista Júlio de Mesquita Filho, Jaboticabal, São Paulo , Brazil ABSTRACT The objective of this experiment was to determine the normal values of bone radiographic density (BRD) by using the optical densitometry in radiographic images and the biochemical values represented by serum calcium, ash percentage, and minerals (calcium, phosphorus, and magnesium) from tibia ash of Cobb broilers at 8, 22, and 43 d of age. A total of 14 broilers were used for densitometric analysis, and 15 were used for biochemical dosages. The BRD values increased (P < 0.05) with age and in all tibia regions (proximal epiphysis, diaphysis, and distal epiphysis), concluding that growth was a determinative factor for bone performance, demanding a higher BRD during broiler development. Tibia proximal epiphysis presented higher BRD values in relation to the other bone regions (P < 0.05), as a result of a possible biomechanical adaptation to ligaments and tension of the muscle tendons at this region, allowing the support of the muscle mass increase. The serum calcium values were kept constant, as a result of the appropriate nutritional levels of the diet that supported the animal homeostasis. The bone ash and mineral percentage increased (P < 0.05) at 22 d of age, due to the higher mineral requirement in this age. The correlation between bone densitometry and the invasive techniques showed that the bone densitometry can substitute the determination of mineral percentage in the ash. This experiment presented normal values of the noninvasive and invasive methods more used in aviculture, allowing us to compare, subsequently, pathological and physiological values or results of broilers fed with different diets. Key words: bone ash, bone mineral density, broiler, serum calcium, tibia 2009 Poultry Science 88 : doi: /ps INTRODUCTION The bone tissue presents a plasticity that makes it able to answer stimuli, adapting itself to the pathological or physiological conditions that are submitted. The BW increases that occur with age (Robson et al., 2006) and the skeletal system diseases, such as rachitism (Mendonça Júnior, 2000) and tibial dyschondroplasia (Almeida Paz et al., 2004), are stimuli that can result in bone structure alterations and bone radiographic density (BRD) variations. The variations in the diet electrolytic balance can influence the acid-base balance, and this fact can endanger the bone development of the broilers (Oliveira et al., 2003). Appropriate nutritional levels promote the normal development of bone tissue (Almeida Paz and Bruno, 2006); therefore, nutritional deficiencies can cause BRD alterations. Currently, there are 2 main research concerns related to skeletal disorders in poultry: osteoporosis in egg-laying hens and leg 2009 Poultry Science Association Inc. Received February 20, Accepted August 16, Corresponding author: barreiro_vet@yahoo.com.br disorders caused by rapid bone growth in broiler chickens (Fleming, 2008). In aviculture, the BRD variations have been measured by invasive techniques, such as determination of the bone ash percentage and analysis of bone minerals (Schreiweis et al., 2003), but to do these techniques, it is necessary to euthanize the animal, which represents a loss to the producer. Bone densitometry can be used in live poultry as a tool to measure bone integrity with similar precision to other invasive techniques (Schreiweis et al., 2003); however, bone densitometry is not widely accepted because the standardization is difficult (Louzada et al., 2001; Almeida Paz et al., 2004). This absence of radiographic standardization is surpassed when densitometric references are used, for example, aluminum stairs, which pass through all the phases of the radiographic process (Louzada et al., 2001) correcting possible alterations that can occur during the image processing. The hydroxyapatite present in the bone structure and the aluminum have a similar density, making it possible to relate the different degrees of bone mineralization with the aluminum amount in the stairs by using the radiographic technique (Loubel and Dubois, 1973). Bone densitometry is efficient in the 2644

2 BONE MINERALIZATION IN BROILERS 2645 accompaniment of diseases that affect the bone tissue because the injured region presents a higher BRD value during the new bone tissue deposition. This increase in the BRD values is due to mineralization of dead osteocytes (Cruess and Dumont, 1985; Pharr and Bargai, 1997). Bone densitometry is an efficient indicator of the tibia ash percentage in broilers, and the correlation between these techniques is 86% (Onyango et al., 2003). The BRD can also be determined by the bone mineral composition (Almeida Paz and Bruno, 2006), taking the measures of calcium and phosphorus present in bone ash (Kienzle et al., 1995), and also determining the bone ash amount (Grier et al., 1996). The bird skeleton is constituted of 99% calcium reserve and 90% corporal phosphorus (Mendonça Júnior, 2000). In the growth phase, the majority of the calcium is used for the bone formation, allowing the support of the BW (Silva et al., 2001). The phosphorus deposition in the bone follows the calcium deposition, characterizing the interdependence between these minerals (Dell Isola et al., 2003). It is difficult to have access to magnesium amount in live animals because this plasmatic ion represents only 1% of total magnesium (Elin, 1987; Seiler, 1990; Blanchflower and Kennedy, 1991; Altura et al., 1994; Anderson and Talcott, 1994; Ising et al., 1995). Under deficiency conditions, the bone magnesium can be a plasmatic magnesium source (Rosol and Capen, 1997). Ion removal results in exchange of the magnesium for calcium on the bone surface. Therefore, the calcium-magnesium relation in ashes is the most sensible indicator of magnesium deficiency (Blaxter and Sharman, 1955). The objective of this experiment was to determine the normal values of BRD by using the optical densitometry in radiographic images and the biochemical values corresponding to serum calcium, ash percentage, and the minerals (calcium, phosphorus, and magnesium) from tibia ash of broilers at different ages (8, 22, and 43 d of age). The experiment offers results that can be used in other experiments of broilers fed with different nutritional levels, and they can also be related to pathological values, allowing the diagnosis of diseases that affect the integrity of the poultry leg. MATERIALS AND METHODS A total of 29 Cobb male broilers were used. They were housed from 1 d of age and bred for 43 d. The broilers were distributed in 2 boxes: one box containing 14 birds, used for densitometric measures, and the other box containing 15 birds, used for biochemical analysis of tibia. The handling was the same as that used in the commercial breed of broilers. The birds were vaccinated at 7 d of age against Gumboro and Newcastle diseases and against Gumboro disease at 14 d of age (reinforcement). The water and ration were provided ad libitum and the diet was done with soybean meal and corn base, in agreement with Rostagno et al. (2005; Table 1). Vitamins A and E were acquired from Sanofi-Aventis (Paris, France). Vitamin E activity was 52%. Densitometric Analysis The densitometric evaluations were realized at 8, 22, and 43 d of age from radiographic images. A total of 14 broilers were positioned dorsal decubitus, and the radiographic images taken followed the technique developed by Louzada (1994). An aluminum staircase composed of 12 steps was used (metallic alloy 6063, in accordance with the Brazilian Association of Technique Norms). The first step measured 0.5 mm in height, increasing 0.5 mm with each step until the tenth. The 11th step measured 6.0 mm, the 12th step measured 0.8 mm, and the step area was 5 25 mm 2. The images from tibia and aluminum stairs were obtained together, in the same radiography. The x-ray equipment (Tridoro 812E, Siemens, São Paulo, Brazil) of the radiology department of veterinary hospital at Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista, (Jaboticabal, São Paulo, Brazil) was adjusted for 8, 22, and 43 d of age, and the following values of kilovoltage, milliamperage, and milliamperage second were used for the respective ages: 35 kv, 100 ma, and 5 mas; 38 kv, 200 ma, and 4 mas; and 40 kv, 200 ma, and 4 mas. A focus-to-film distance of 1 m was used for all radiographic images taken. Kodak films (T-MATG/ RA, Kodak, Manaus, Brazil) were used inside the cassettes and measured 18 24, 24 30, and cm. The revelation and fixation of the films were made by automatic process, using Kodak equipment (X-OMAT 2000, Kodak). The radiographic images were scanned by a Scaníon scanner (Express A3 USB, Íon Indústria, São Paulo, Brazil) and these images were submitted to the computer program Image-Pro Plus (version 4.1, Media Cybernetics, Silver Spring, MD), and the density calibration was made, having the aluminum stairs image as reference. Subsequently, the BRD from left tibia was measured at the different regions, (proximal epiphysis, diaphysis, and distal epiphysis) and at the different ages. The densitometric values were expressed in aluminum millimeters. Biochemical Analysis Three milliliters of blood was collected from the jugular vein from 5 broilers at 8, 22, and 43 d of age. Subsequently, the samples were centrifuged and the serum was used for the calcium dosage. These same birds mentioned above were killed by cervical dislocation, which is a euthanasia method accepted by the Ethics and Animal Welfare Commission of Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista. The left tibias were obtained after the removal of skin, muscles, ligaments, vessels, and epi-

3 2646 Barreiro et al. Table 1. Composition and nutritive values of experimental diets Item Initial (1 to 7 d) Growth (8 to 21 d) Final (22 to 43 d) Ingredients (%) Ground corn Soybean meal Soybean oil Dicalcium phosphate Limestone Vitamin-mineral premix Salt dl-methionine l-lysine-hcl Calculated composition (%) ME (Mcal/kg) CP Calcium Total phosphorus Available phosphorus Sodium Total lysine Available lysine Total methionine Available methionine Total methionine + cystine Available methionine + cystine Vitamin-mineral premix supplied the following per kilogram of ration: selenium (0.273 mg), nicotinic acid (34.65 mg), copper (125 mg), calcium pantothenate (9.5 mg), biotin (0.16 mg), manganese (0.076 mg), dl-methionine (1.7 g), iodine (1.3 mg), coccidiostat (125 mg), antioxidant (0.5 mg), choline (0.6 mg), vitamin A (7,000 IU), vitamin B 1 (1.78 mg), vitamin B 12 (0.01 mg), vitamin B 2 (9.6 mg), vitamin B 6 (3.46 mg), vitamin D 3 (3,000 IU), vitamin E (25 IU), vitamin K (0.98 mg), and zinc (0.091 mg). The additives were added in the inert space. physary cartilage. The bones were placed in an oven (55 C) for 72 h to dry them. Subsequently, these bones were grinded in a ball mill and put in mufla oven at 550 C for 3 h, and the ash was used to quantify calcium, phosphorus, and magnesium amounts in the bone, by the technique of Silva and Queiroz (2002). The calcium and magnesium values were determined by atomic absorption in GBC equipment (932AA, Analitica, São Paulo, Brazil), whereas the phosphorus determination was realized by a spectrophotometer (B-395, Micronal, São Paulo, Brazil). The serum calcium was quantified by Calcium Liquiform Kit (Labtest Diagnóstica S.A., Lagoa Santa, Brazil). Statistical Analysis Repeated measures ANOVA was the statistical method used to evaluate the BRD values, which were obtained from live broilers, with a factor region among the broilers with 3 levels (proximal epiphysis, diaphysis, and distal epiphysis) and a factor age with 3 levels (8, 22, and 43 d of age) within the broilers in a randomized block design. There were n = 14 broilers (blocks). Single factor ANOVA was the statistical method used to evaluate the biochemical dosages, which were obtained from killed broilers. It was compared in 3 treatments (ages) with 5 replications (5 broilers) for each age. Pair-wise comparisons of means were made using Tukey s test procedure. A P-value <0.05 was considered significant. All of the statistical results were obtained from Minitab program (model 14, Minitab Inc., State College, PA). RESULTS AND DISCUSSION The technique of bone densitometry in radiographic images was used in this experiment because it has easy access, low cost, reliable results, and this technique was used with success by other authors, such as Campos (2005), Godoy et al. (2005), Almeida Paz et al. (2006), and Fleming (2008). The densitometric values (Table 2) have demonstrated that the BRD increased with age (8, 22, and 43 d of age) and in all tibia regions (proximal epiphysis, diaphysis, and distal epiphysis), presenting a significant interaction between age and region. This fact has evidenced a tibia mineralization that allows the support of the weight increase during its growth. The BRD values at 8, 22, and 43 d of age in the different regions of the tibia have presented significant differences. The proximal epiphysis has showed higher BRD values than diaphysis and distal epiphysis, probably as result of a biomechanical adaptation that the Table 2. Values (mean ± SD) of the bone radiographic density (aluminum millimeters) from proximal epiphysis, diaphysis, and distal epiphysis of the tibia of broilers at 8, 22, and 43 d of age Age Proximal epiphysis Diaphysis Distal epiphysis ± 0.47 a,z 1.88 ± 0.39 b,z 1.42 ± 0.39 c,z ± 0.58 a,y 3.34 ± 0.36 b,y 1.96 ± 0.30 c,y ± 0.67 a,x 5.04 ± 0.31 b,x 3.43 ± 0.43 c,x a c Means within a row lacking a common superscript differ (P < 0.05). x z Means within a column lacking a common superscript differ (P < 0.05).

4 Table 3. Values (mean ± SD) of the biochemical analysis represented by serum calcium, tibia ash, and minerals in the ash (calcium, phosphorus, and magnesium) of broilers at 8, 22, and 43 d of age Age Serum calcium (mg/dl) Ash (%) Calcium ash (%) Phosphorus ash (%) Magnesium ash (%) ± 1.97 a ± 3.49 b ± 1.92 b ± 0.73 b 0.44 ± 0.05 b ± 0.72 a ± 2.02 a ± 1.59 a ± 0.85 a 0.63 ± 0.04 a ± 1.68 a ± 1.53 b ± 0.83 a ± 0.71 a 0.58 ± 0.09 a a,b Means within a column lacking a common superscript differ (P < 0.05). BONE MINERALIZATION IN BROILERS 2647 proximal epiphysis suffers due to ligament action and the muscle tendon tension of long fibular, short fibular, gastrocnemius, digital extensor, and popliteum muscles that originate or are inserted at this location. This biomechanical adaptation allows the support by the tibia of the heaviest muscles during broiler growth. Robson et al. (2006) have observed that BW has a strong relation with the BRD values. A variation of the electrolytic balance can influence the acid-basic balance, modifying the serum ph, and changes in this balance can endanger the bone development in poultry because the maintenance of the blood acid-basic balance is directly connected to the bone mineralization process (Oliveira et al., 2003). The serum calcium values (Table 3) did not present significant variations with age. This stability on values was expected, considering that the broilers were fed with balanced rations during the experimental period, attending the conditions to the animal homeostasis maintenance. The BRD can be determined using the ash percentage, which is an invasive method, and that can be substituted to a noninvasive method, such as bone densitometry in radiographic images. The disadvantage of the invasive methods is the necessity of poultry euthanasia, which represents a loss to the producer. Onyango et al. (2003) have concluded that bone densitometry presents a correlation of 86% with ash percentage, which indicates that bone densitometry is an efficient method to measure bone mineralization. It was possible to verify in this experiment that the bone ash percentage increased at 22 d of age as a function of the higher mineral exigency in this phase (growth phase) (Table 3). The balance between calcium and phosphorus is of utmost importance for leg health in broilers (Fleming, 2008). The calcium, phosphorus, and magnesium levels in ash increased significantly at 22 d of age and similar values were found at 43 d of age. Schoulten et al. (2003) have found values between 54.8 and 56.5% of tibia ash, 18.8 and 20.1% of calcium in ash, and 10.4 and 11.9% of phosphorus in ash from Hubbard-MPK broilers at 42 d of age. Oliveira et al. (2003) have compared diet efficiency and have determined the following values: 17.86% of tibia ash, 5.61% of calcium in ash, and 3.27% of phosphorus in ash from Cobb-Vantress broilers at 21 d of age fed with viscera flour and 17.82% of tibia ash, 5.42% calcium in ash, and 3.17% of phosphorus in ash from broilers fed with feather flour. Silva et al. (2001) have found values corresponding to 30.66% of tibia ash, 37.53% of calcium in ash, 21.36% of phosphorus in ash, and 0.95% of magnesium in ash from Hubbard broilers at 14 d of age. The ash percentage found by Schoulten et al. (2003) was higher than the values found in this experiment, although the mineral percentage was lower, considering that the ash analysis was done at 42 d of age. Oliveira et al. (2003) have found mineral percentage in ash (calcium and phosphorus) and ash percentage to be very low when the results were compared with these experiment values, indicating that a lower tibia mineralization has occurred. Silva et al. (2001) have showed calcium and phosphorus values close to this experiment, but the magnesium values were a little higher. Onyango et al. (2003) have concluded that bone densitometry is an efficient method to measure bone mineralization. The correlation between bone densitometry and the invasive techniques (Table 4) has presented significant differences for the values of minerals (calcium, phosphorus, and magnesium) in the ash, evidencing that the noninvasive technique of bone densitometry can substitute the determination of mineral percentage in the ash. In conclusion, broiler growth was a determinative factor to tibia development, increasing the exigencies in relation to BRD values over the course of ages. There was also a possible biomechanical adaptation of tibia proximal epiphysis to ligaments, muscles, and tendon tension, allowing the muscle mass support that happens during its growth. There were no oscillations in the serum calcium levels due to the appropriate nutrient levels in the diet that have kept the animal homeostasis. The growth and final phases have demanded a higher mineral exigency to the tibia, leading to increased mineral values in ash and the ash percentage at Table 4. Correlation values between bone densitometry and the invasive measurements (serum calcium, ash content, and minerals in the ash) Item Serum calcium Ash Calcium ash Phosphorus ash Magnesium ash Correlation P-value

5 d of age. The correlation between bone densitometry and the invasive techniques has showed that the bone densitometry can substitute the determination of mineral percentage in the ash. ACKNOWLEDGMENTS This research was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), São Paulo, Brazil. REFERENCES Almeida Paz, I. C. L., and L. D. G. Bruno Bone mineral density: Review. Braz. J. Poult. Sci. 8: Almeida Paz, I. C. L., A. A. Mendes, R. R. Quinterio, L. C. Vulcano, S. E. Takahashi, R. G. Garcia, C. M. Komiyama, A. Balog, K. Pelicia, F. S. Wescheler, P. S. O. Scudeller, and A. Piccinin Bone mineral density of tibia and femur of broiler breeders: Growth, development and production. Braz. J. Poult. Sci. 8: Almeida Paz, I. C. L., A. A. Mendes, T. S. Takita, L. C. Vulcano, P. C. Guerra, F. S. Wechsler, and R. G. Garcia Tibial dyschondroplasia and bone mineral density. Braz. J. Poult. Sci. 6: Altura, B. T., T. L. Shirey, C. C. Young, K. Dell Orfano, J. Hiti, R. 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