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Effects of age, vitamin D3, and fructooligosaccharides on bone growth and skeletal integrity of broiler chicks
Effects of age, vitamin D3, and fructooligosaccharides on bone growth and skeletal integrity of broiler chicks W. K. Kim,*1 S. A. Bloomfield,† and S. C. Ricke‡ *Department of Animal Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; †Department of Health and Kinesiology, Texas A & M University, College Station 77843; and ‡Center for Food Safety and Department of Food Science, University of Arkansas, Fayetteville 72704 (P = 0.016, 0.0003, 0.0002, 0.01, and 0.0001, respectively). Total, cortical, and trabecular BMD of chick proximal femurs were not influenced by age. However, BMC and bone area were significantly affected by age. The femurs of 2-wk-old chicks exhibited significantly lower stiffness and ultimate load than those of 3-wk-old chicks (P = 0.0001), whereas ultimate stress and elastic modulus of the femurs of 2-wk-old chicks were significantly higher than that of femurs of 3-wk-old chicks (P = 0.0001). Chicks fed 250 µg/kg of vitamin D3 exhibited significantly greater midshaft cortical BMC (P = 0.04), bone area (P = 0.04), and thickness (P = 0.03) than control 2, 2% FOS, or 4% FOS chicks. In summary, our study suggests that high levels of vitamin D3 can increase bone growth and mineral deposition in broiler chicks. However, FOS did not have any beneficial effects on bone growth and skeletal integrity. Age is an important factor influencing skeletal integrity and mechanical properties in broiler chicks.
Key words: age, vitamin D, fructooligosaccharide, bone mineral density, mechanical property 2011 Poultry Science 90:2425–2432 doi:10.3382/ps.2011-01475
INTRODUCTION High growth rates in animals are generally associated with musculoskeletal weakness because of the development of bone that is much less organized, less dense, and more porous than the bones of slow-growing animals (Williams et al., 2004; Bennett, 2008). Rapid muscle growth in broiler chicks (meat-type chicks) through genetic selection causes imbalance between meat production and skeletal growth, resulting in abnormal skeletal development (Oviedo-Rondon et al., 2006). Leg problems and skeletal disorders, such as lameness, twisted legs, tibial dyschondroplasia (TD), and crooked toes, are the common causes of culling and mortality in broiler chicks (Bradshaw et al., 2002; Oviedo-Rondon ©2011 Poultry Science Association Inc. Received March 10, 2011. Accepted July 24, 2011. 1 Corresponding author: [email protected]
et al., 2006). Because leg problems in broilers cause pain, limit their mobility to access feed and water, and result in economic losses estimated at $120 million/yr (Cook, 2000), these are important animal welfare and economic issues (Oviedo-Rondon et al., 2009). One strategy that can be used to prevent leg problems and skeletal disorders is stimulating bone growth and strength in the early growth period. One potential nutrient or bioactive molecule is vitamin D3. Vitamin D3 is considered to be a prohormone generated in the skin through UV irradiation of 7-dehydrocholesterol or absorbed from the diet in the intestinal tract (DeLuca, 2004). It is not biologically active in this initial form and needs to be converted to 25-hydroxyvitamin D3 in the liver and subsequently to 1,25-dihydroxyvitamin D3 in the kidney (DeLuca, 2004). 1,25-Dihydroxyvitamin D3 acts through a nuclear receptor to express its numerous functions, including bone homeostasis, Ca, and phosphate absorption in the small intestine, Ca mobilization in bone, and Ca reabsorption in the kid-
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ABSTRACT A study was conducted to evaluate the effects of age, vitamin D3, and fructooligosaccharides (FOS) on bone mineral density (BMD), bone mineral content (BMC), cortical thickness, cortical and trabecular area, and mechanical properties in broiler chicks using peripheral quantitative computed tomography and mechanical testing. A total of 54 male broiler chicks (1 d old) were placed in battery brooders and fed a corn–soybean starter diet for 7 d. After 7 d, the chicks were randomly assigned to pens of 3 birds each. Each treatment was replicated 3 times. There were 6 treatments: 1) early age control (control 1); 2) control 2; 3) 125 µg/kg of vitamin D3; 4) 250 µg/kg of vitamin D3; 5) 2% FOS); and 6) 4% FOS. The control 1 chicks were fed a control broiler diet and killed on d 14 to collect femurs for bone analyses. The remaining groups were killed on d 21. Femurs from 3-wk-old chicks showed greater midshaft cortical BMD, BMC, bone area, thickness, and marrow area than those from 2-wk-old chicks
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bone growth and skeletal integrity in broiler chicks and that pQCT and mechanical testing would be excellent tools for evaluating bone quality and mechanical properties in broiler chicks. Therefore, the objective of this study was to evaluate the effects of age, vitamin D3, and FOS on BMD, BMC, cortical thickness, cortical area, marrow area, and mechanical properties in broiler chicks by pQCT and mechanical testing.
MATERIALS AND METHODS General Procedures A total of 54 male broiler chicks (1 d old) were placed in heated Petersime battery brooders (Petersime Incubator Co., Gettysburg, OH) and fed a corn–soybean broiler starter diet for 7 d. The starter diet contained 23% CP, 3,200 kcal/kg of ME, 1% Ca, 0.45% available P, and 90 µg/kg of vitamin D3. The chicks were individually weighed and then randomly assigned to pens of 3 birds each. Each treatment was replicated 3 times. There were 6 treatments: 1) early age control (control 1); 2) control 2; 3) 125 µg/kg of vitamin D3; 4) 250 µg/ kg of vitamin D3; 5) 2% FOS; and 6) 4% FOS. Vitamin D3 and FOS were purchased from Sigma-Aldrich (St. Louis, MO). The control 1 chicks were fed a control broiler diet and killed by cervical dislocation on d 14. The chicks from the other groups were fed a control broiler diet and treatment diets containing 125 µg/kg of vitamin D3, 250 µg/kg of vitamin D3, 2% FOS, or 4% FOS and were killed by cervical dislocation on d 21. For vitamin D3 treatments, 125 or 250 µg/kg of vitamin D3 was added to the control diet containing 90 µg/kg of vitamin D3. Left femurs were collected for BMD, BMC, bone area, bone thickness, and mechanical testing. The bones were cleaned of attached tissue, wrapped in PBS-soaked gauze for pQCT and mechanical testing, and stored at −20°C. All animal procedures were approved by the Texas A & M University Lab Animal Care Committee.
pQCT Bone scans of left femurs were performed with an XCT Research instrument (Stratec, Norland, Fort Atkinson, WI). This model has a minimum voxel size of 0.07 mm and a scanning beam thickness of 0.50 mm. Scan sites included the midshaft femurs (3 slices of each bone located at one-half the total bone length ± 2 mm) for volumetric BMD of the cortical bones; the proximal femurs were scanned to assess metaphyseal trabecular volumetric BMD (3 slices located 8.0, 8.5, and 9.0 mm from the proximal end of the bone). All scans were obtained at a scan speed of 2.5 mm/s, with a voxel resolution of 0.07 × 0.07 × 0.50 mm. In addition, the middiaphyseal cross-sectional moment of inertia (CSMI), which is a measure of bone material in a cross-section area and indicates its distribution around the neutral bending axis to resist bending (Bennett, 2008), was ob-
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ney (Farquharson et al., 1993; Leeson and Summers, 2001; DeLuca, 2004). It has been reported that the supplementation of high levels of vitamin D3 reduced incidence of leg problem, such as TD and Ca rickets in broilers (Whitehead et al., 2004; Atencio et al., 2005; Driver et al., 2006). However, few studies have used peripheral quantitative computed tomography (pQCT) and mechanical testing to evaluate whether high levels of vitamin D3 stimulate bone growth and enhance skeletal integrity in young broilers. Another potential bioactive molecule is fructooligosaccharide (FOS). Fructooligosaccharides are low molecular weight, nondigestible carbohydrates that have previously been examined as prebiotics in laying hens for their ability to limit Salmonella Enteritidis colonization and their influence on bone metabolism (Kim et al., 2006; Donalson et al., 2007, 2008a,b). Fructooligosaccharides also have the potential to increase bone formation and reduce bone resorption (Ohta et al., 1998; Takahara et al., 2000). Several studies have demonstrated that FOS enhanced true Ca, Mg, Fe, and P absorptions in the intestine of rats (Ohta et al., 1993, 1995; Kashimura et al., 1996; Morohashi et al., 1998). Moreover, FOS compounds have been shown to increase Ca balance and bone mineral density (BMD) in growing rats (Ohta et al., 1998). Takahara et al. (2000) observed that FOS increased femoral bone volume and mineral concentrations in rats. It has been shown that FOS induced intestinal Ca-binding protein (calbindin-D9k) levels in rats (Takasaki et al., 2000). More recently, Zafar et al. (2004) reported that FOS increased femoral Ca content and BMD whereas FOS reduced bone resorption rate in ovariectomized rats. However, no research has been conducted to evaluate the effects of FOS on bone growth and Ca metabolism in broiler chicks. Recently, dual-energy X-ray absorptiometry (DEXA) has been used to measure bone density and mineral content in broiler chicks and laying hens (Kim et al., 2004; Talaty et al., 2009). It has been demonstrated that BMD and bone mineral content (BMC) obtained by DEXA are positively correlated with bonebreaking force, bone ash, bone ash concentration, and bone weight in chickens (Mazzuco and Hester, 2005; Kim et al., 2006, 2008). Bone mineral density and BMC are highly correlated with each other and are good indicators for bone strength. However, few studies have evaluated bone quality in broiler chicks using pQCT, which can serve as a noninvasive method for measuring BMD, BMC, cross-section area, and 3-dimensional visualization of the bone (Clark et al., 2005). Because pQCT measures true volumetric bone density in specific sites (Korver et al., 2004; Kim et al., 2007), it can offer more precise and detailed information about site-specific BMD and cross-sectional geometry than DEXA. Thus, in the present study, we measured BMD and BMC in broiler chicks using pQCT. We hypothesized that high levels of vitamin D3 and FOS supplementation in broiler diets would increase
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tained with respect to the neutral bending axis of the femur bone shaft during 3-point bending (mechanical property testing).
Mechanical Testing
E = S × L3/(48,000 × CSMI), and
Effects of Age on Cortical BMD, BMC, Bone Area, Bone Thickness, and Mechanical Properties First, we evaluated the effects of age on bone growth and skeletal integrity of chick midshaft femurs (Table 1). Three-week-old chicks (control 2) had significantly higher BMD (P = 0.016) and BMC (P = 0.0003) compared with 2-wk-old chicks (control 1; Table 1). Femurs from 3-wk old chicks also showed greater midshaft cortical bone area (P = 0.0002), thickness (P = 0.01), and marrow area (P = 0.0001) than those from 2-wkold chicks. Next, we evaluated the effects of age on total, cortical, and trabecular BMD, BMC, and bone area of chick proximal femurs (Figure 1). Total, cortical, and trabecular BMD of chick proximal femurs were not influenced by age. However, BMC and bone area were significantly affected by age. Total, cortical, and trabecular BMC of control 2 chicks (3 wk old) were significantly higher than that of control 1 chicks (2-wk old; P = 0.0002, 0.04, and 0.003, respectively). Total and cortical bone areas and marrow area of control 2 chicks were also significantly larger than that of control 1 chicks (P = 0.0003, 0.01, and 0.0007, respectively). We further measured mechanical properties of chick femurs (Figure 2). The femurs of 2-wk-old chicks (control 1) exhibited significantly lower stiffness and ultimate load (structural properties) than those of 3-wkold chicks (control 2; Figure 2A and B; P = 0.0001), whereas ultimate stress and elastic modulus (material properties) of the femurs of 2-wk-old chicks were significantly higher than those of 3-wk-old chicks (Figure 2C and D; P = 0.0001). These results indicate that age is an important factor for bone growth and skeletal integrity in broiler chicks.
Effects of Vitamin D3 and FOS on BMD, BMC, Bone Area, Bone Thickness, and Mechanical Properties
US = UL × L × D/(8 × CSMI).
Statistical Analysis All data were subjected to 1-way ANOVA as a completely randomized design using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). Significant differences among the means were determined using Duncan’s multiple-range test at P < 0.05.
We evaluated the effects of vitamin D3 and FOS on bone growth and skeletal integrity in midshaft cortical bone of chick femurs (Figure 3). No significant differences were found in midshaft cortical BMD among the treatments (Figure 3A; P = 0.8306). However, chicks fed 250 µg/kg of vitamin D3 exhibited significantly
Table 1. Effects of broiler age on midshaft cortical bone mineral density (BMD), bone mineral content (BMC), bone area, bone thickness, and marrow area of femurs (n = 3/treatment) Treatment1
BMD (mg/cm3)
BMC (mg/mm)
Bone area (mm2)
Bone thickness (mm)
Marrow area (mm2)
Control 1 Control 2 Pooled SE P-value
692b 744a 8.5 0.016
9.2b 17.9a 0.51 0.0003
13.3b 24.1a 0.55 0.0002
1.33b 1.62a 0.04 0.01
8.02b 15.92a 0.17 0.0001
a,bMeans 1Control
within a column with different superscripts differ significantly (P < 0.05). 1 = 2-wk-old chicks; control 2 = 3-wk-old chicks.
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After pQCT scans were completed, mechanical properties of the midshaft femurs were determined by 3-point bending to failure with an Instron 1125 servo-controlled testing machine (Instron Corp., Canton, MA) according to previously published procedures (Allen and Bloomfield, 2003). The bones were thawed at room temperature and placed posterior side down on metal pin supports located ±10 mm (femurs) from the middiaphysis testing site. With a 455-kg load cell, quasistatic loading (2.5 mm/min) was applied to the anterior surface of the femurs until fractures occurred. All specimens were sprayed with PBS just before testing to maintain hydration. Displacements were monitored by a linear variable differential transformer interfaced with a personal computer. Raw data, collected at 10 Hz as load versus displacement curves, were analyzed with Table-Curve 2.0 software (Jandel, San Rafael, CA). Structural variables (ultimate load and stiffness) were obtained directly from the load:displacement curves. The maximum load obtained was defined as the ultimate load (UL, in N), and the slope of the elastic portion of the curve was defined as stiffness (S, in N/mm). Bone tissue material properties were calculated by normalizing structural properties for cross-sectional bone geometry at the site of testing by using CSMI (in mm4 from pQCT), bone diameter (D, in mm) as measured by calipers, and the appropriate bottom support span distance (L, which was 20 or 60 mm). The appropriate formulas for elastic modulus (E, in GPa) and ultimate stress (US, in MPa) were as follows:
RESULTS
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Figure 1. Effect of age on total, cortical, and trabecular bone mineral density (BMD), bone mineral content (BMC), and bone area of proximal femurs (n = 3/treatment). Control 1 = 2-wk-old chicks (early age control); control 2 = 3-wk-old chicks. Means with different letters differ significantly (P < 0.05).
higher midshaft cortical BMC than control 2, 2% FOS, or 4% FOS chicks (Figure 3B; P = 0.04). Furthermore, chicks fed 250 µg/kg of vitamin D3 had significantly larger midshaft cortical bone area and thickness compared with control 2, 2% FOS, or 4% FOS chicks (Figure 3C and D; P = 0.04 and 0.03, respectively). Bone marrow area of chicks fed 250 µg/kg of vitamin D3 was
significantly larger than that of control 2, 2% FOS, or 4% FOS chicks (Figure 3E; P = 0.01). In the present study, no significant differences were found in feed intake and weight gain among the treatment groups (data not shown; P = 0.643 and 0.478, respectively). We also determined total, cortical, trabecular BMD, BMC, and area and mechanical properties of proximal
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cal BMC than those fed 125 µg/kg of vitamin D3 or 2% FOS (Figure 5; P = 0.01). However, no significant differences were found in total, cortical, and bone marrow area among the treatments (Figure 6; P = 0.2676, 0.107, and 0.3325, respectively). We also evaluated the effects of vitamin D3 and FOS on mechanical properties. However, no significant differences were found in all mechanical properties (stiffness, ultimate load, ultimate stress, and elastic modulus) among the treatments (data not shown; P = 0.533, 0.949, 0.086, and 0.479, respectively).
DISCUSSION
femurs (Figures 4, 5, and 6). Total density of chicks fed 250 µg/kg of vitamin D3 was significantly higher than that of those fed 125 µg/kg of vitamin D3 or 2% FOS (Figure 4; P = 0.0487). However, no significant differences were found in cortical and trabecular BMD of proximal femurs among the treatments (Figure 4; P = 0.279 and 0.601, respectively). Chicks fed 250 µg/kg of vitamin D3 had significantly higher total and corti-
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Figure 2. Effect of age on A) stiffness, B) ultimate load, C) ultimate stress, and D) elastic modulus of chick femurs (n = 3/treatment). Control 1 = 2-wk-old chicks (early age control); control 2 = 3-wk-old chicks. Means with different letters differ significantly (P < 0.05).
In this study, we found that 3-wk-old chicks exhibited significantly greater midshaft cortical BMD, BMC, bone area, bone thickness, and marrow area than 2-wkold chicks, indicating that age is an important factor for bone growth and skeletal integrity. This result also indicates that endocortical resorption by osteoclasts (bone resorbing cells) occurs on the endosteal surface (inner edge of cortical shell) to increase marrow area while new bone formation by osteoblasts (bone forming cells) occurs on the periosteal surface (outside of cortical shell) to increase cortical bone area in broiler chicks as chick age increases. This is called radial or periosteal expansion (Bain and Watkins, 1993) and is a way to increase bone size and bone marrow area in broiler chicks as chick age increases. Interestingly, unlike midshaft femurs, total, cortical, and trabecular BMD values of proximal femurs were not influenced by age, whereas BMC and bone area of proximal femurs from 3-wk-old chicks were significantly higher than that of 2-wk-old chicks. These results suggest that bone density in specific sites of bones may be differently influenced by age. We also found that stiffness and ultimate load of femurs increased whereas ultimate stress and elastic modulus decreased as chick age increased. It appears that stiffness and ultimate load are negatively related to ultimate stress and elastic modulus in broiler chicks. Age-dependent changes of mechanical properties have been suggested by other researchers (Lawrence et al., 1994; Baumer et al., 2009). Our mechanical test showed that stiffness and ultimate load of chick femurs increased but ultimate stress and elastic modulus decreased as chick age increased. Because stiffness is important for effective locomotion (Kjaer, 2004), stiffer bones in older chicks might help improve the efficiency of leg movements. Furthermore, rapid growth in broiler chicks significantly reduces ultimate stress and elastic modulus independent of the bone size that confers increased stiffness and ultimate load. These results are in agreement with a swine study conducted by Baumer et al. (2009). Stiffness of the infant porcine parietal bone has been demonstrated to increase with age, whereas ultimate stress was reduced with age (Baumer et al., 2009). The results in the current study suggest that mechanical properties in broiler chicks are influenced by chick age.
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Figure 3. Midshaft cortical bone mineral density (BMD), bone mineral content (BMC), bone area, and bone thickness of femurs from 3-wk-old chicks (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides. Means with different letters differ significantly (P < 0.05).
In the present study, although no significant differences were found in midshaft cortical BMD among the treatments, chicks fed 250 µg/kg of vitamin D3 had significantly higher midshaft cortical BMC and larger bone area, bone thickness, and marrow area compared
with control. These results indicate that 250 µg/kg of vitamin D3 stimulates mineral deposition and radial growth of midshaft cortical bone in broilers. Increased rates of mineral deposition and radial growth by 250 µg/kg of vitamin D3 were well balanced in midshaft
BONE GROWTH AND SKELETAL INTEGRITY OF BROILER CHICKS
femurs to maintain similar BMD values to those of control. However, 250 µg/kg of vitamin D3 treatment did not change any mechanical properties. It also has been shown that chicks fed 250 µg/kg of vitamin D3 had significantly lower incidence of TD (Whitehead et al., 2004), a metabolic skeletal disorder in fast-growing meat-type poultry such as broilers and turkeys (Rath et al., 2004). Stimulating bone growth by high levels of vitamin D3 may be beneficial in reducing incidence of TD. However, FOS treatments did not affect any of the bone parameters. Although several studies have
Figure 6. Total, cortical, and marrow area of proximal femurs (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wkold chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/ kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides.
reported that FOS compounds increase BMD, BMC, and bone volume in rats (Ohta et al., 1998; Takahara et al., 2000; Zafar et al., 2004), FOS treatments did not increase BMC, BMD, bone area, or bone thickness in chicks. Species differential effects of FOS on bone and mineral metabolisms may exist. In summary, our study suggests that high levels of vitamin D3 can increase bone growth and mineral deposition in broiler chicks. However, FOS did not have any beneficial effects on bone growth and skeletal integrity. To reduce incidence of skeletal disorders, high levels of vitamin D3 dietary amendment may be a solution. Because chicks possess a high tolerance for vitamin D3, dietary supplementation of vitamin D3 higher than 250 µg/kg needs to be investigated to further confirm the potential for increases in bone growth and mineral contents, which in turn should lead to the reduction of skeletal problems in broiler chicks. Age is an important factor influencing skeletal integrity and mechanical properties in broiler chicks.
REFERENCES
Figure 5. Total, cortical, and trabecular bone mineral content (BMC) of proximal femurs (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides. Means with different letters differ significantly (P < 0.05).
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Figure 4. Total, cortical, and trabecular bone mineral density (BMD) of proximal femurs (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides. Means with different letters differ significantly (P < 0.05).
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Kim et al. Korver, D. R., J. L. Saunders-Blades, and K. L. Nadeau. 2004. Assessing bone mineral density in vivo: Quantitative computed tomography. Poult. Sci. 83:222–229. Lawrence, L. A., E. A. Ott, G. J. Miller, P. W. Poulos, G. Piotrowski, and R. L. Asquith. 1994. The mechanical properties of equine third metacarpals as affected by age. J. Anim. Sci. 72:2617–2623. Leeson, S., and J. D. Summers. 2001. Scott’s Nutrition of the Chicken. Publ. Univ. Books, Guelph, Ontario Canada. Mazzuco, H., and P. Y. Hester. 2005. The effect of an induced molt and a second cycle of lay on skeletal integrity of White Leghorns. Poult. Sci. 84:771–781. Morohashi, T., T. Sano, A. Ohta, and S. Yamada. 1998. True calcium absorption in the intestine is enhanced by fructooligosaccharide feeding in rats. J. Nutr. 128:1815–1818. Ohta, A., Y. Motohashi, M. Ohtsuki, M. Hirayama, T. Adachi, and K. Sakuma. 1998. Dietary fructooligosaccharides increase calcium absorption and levels of mucosal calbindin-D9k in the large intestine of gastrectomized rats. Scand. J. Gastroenterol. 33:1062–1068. Ohta, A., M. Ohtsuki, S. Baba, T. Adachi, T. Sakata, and E. Sakaguchi. 1995. Calcium and magnesium absorption from the colon and rectum are increased in rats fed fructooligosaccharides. J. Nutr. 125:2417–2424. Ohta, A., N. Osakabe, K. Yamada, Y. Saito, and H. Hidaka. 1993. Effect of fructooligosaccharides and other saccharides on Ca, Mg and P absorption in rats. J. Jpn. Soc. Nutr. Food Sci. 46:123– 129. Oviedo-Rondon, E. O., P. R. Ferket, and G. B. Havenstein. 2006. Understanding long bone development in broilers and turkeys. Avian Poult. Biol. Rev. 17:77–88. Oviedo-Rondon, E. O., M. J. Wineland, S. Funderburk, J. Small, H. Cutchin, and M. Mann. 2009. Incubation conditions affect leg health in large, high-yield broilers. J. Appl. Poult. Res. 18:640– 646. Rath, N. C., W. E. Huff, J. M. Balog, and G. R. Huff. 2004. Comparative efficacy of different dithiocarbamates to induce tibial dyschondroplasia in poultry. Poult. Sci. 83:266–274. Takahara, S., T. Morohashi, T. Sano, A. Ohta, S. Yamada, and R. Sasa. 2000. Fructooligosaccharide consumption enhances femoral bone volume and mineral concentrations in rats. J. Nutr. 130:1792–1795. Takasaki, M., H. Inaba, A. Ohta, Y. Motohashi, K. Sakai, H. Morris, and K. Sakuma. 2000. Dietary short-chain fructooligosaccharides increase calbindin-D9k levels only in the large intestine in rats independent of dietary calcium deficiency or serum 1,25 dihydroxy vitamin D levels. Int. J. Vitam. Nutr. Res. 70:206–213. Talaty, P. N., M. N. Katanbaf, and P. Y. Hester. 2009. Life cycle changes in bone mineralization and bone size traits of commercial broilers. Poult. Sci. 88:1070–1077. Whitehead, C. C., H. A. McCormack, L. McTeir, and R. H. Fleming. 2004. High vitamin D3 requirements in broilers for bone quality and prevention of tibial dyschondroplasia and interactions with dietary calcium, available phosphorus and vitamin A. Br. Poult. Sci. 45:425–436. Williams, B., D. Waddington, D. H. Murray, and C. Farquharson. 2004. Bone strength during growth: Influence of growth rate on cortical porosity and mineralization. Calcif. Tissue Int. 74:236– 245. Zafar, T. A., C. M. Weaver, Y. Zhao, B. R. Martin, and M. E. Wastney. 2004. Nondigestible oligosaccharides increase calcium absorption and suppress bone resorption in ovariectomized rats. J. Nutr. 134:399–402.
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rietal bone and a correlation to the human. J. Biomech. Eng. 131:111006–111012. Bennett, M. B. 2008. Post-hatch growth and development of the pectoral and pelvic limbs in the black noddy, Anous minutes. Comp. Biochem. Physiol. A 150:159–168. Bradshaw, R. H., R. D. Kirkden, and D. M. Broom. 2002. A review of the aetiology and pathology of leg weakness in broilers in relation to welfare. Avian Poult. Biol. Rev. 13:45–103. Clark, W. D., E. L. Smith, K. A. Linn, J. R. Paul-Murphy, and M. E. Cook. 2005. Use of peripheral quantitative computed tomography to monitor bone healing after radial osteotomy in threeweek-old chickens (Gallus domesticus). J. Avian Med. Surg. 19:198–207. Cook, M. E. 2000. Skeletal deformities and their causes: Introduction. Poult. Sci. 79:982–984. DeLuca, H. F., 2004. Overview of general physiologic features and functions of vitamin D. Am. J. Clin. Nutr. 80:1689S–1696S. Donalson, L. M., W. K. Kim, V. I. Chalova, P. Herrera, J. L. McReynolds, V. G. Gotcheva, D. Vidanović, C. L. Woodward, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2008a. In vitro fermentation response of laying hen cecal bacteria to combinations of fructooligosaccharide (FOS) prebiotic with alfalfa or layer ration. Poult. Sci. 87:1263–1275. Donalson, L. M., W. K. Kim, V. I. Chalova, P. Herrera, C. L. Woodward, J. L. McReynolds, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. In vitro anaerobic incubation of Salmonella enterica serotype Typhimurium and laying hen cecal bacteria in poultry feed substrates and a fructooligosaccharide prebiotic. Anaerobe 13:208–214. Donalson, L. M., J. L. Mc, W. K. Reynolds, V. I. Kim, C. L. Chalova, L. F. Woodward, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2008b. The influence of a fructooligosaccharide prebiotic combined with alfalfa molt diets on the gastrointestinal tract fermentation, Salmonella Enteritidis infection and intestinal shedding in laying hens. Poult. Sci. 87:1253–1262. Driver, J. P., A. Atencio, G. M. Pesti, H. M. Edwards Jr., and R. I. Bakalli. 2006. The effect of maternal dietary vitamin D3 supplementation on performance and tibial dyschondroplasia of broiler chicks. Poult. Sci. 85:39–47. Farquharson, C., C. C. Whitehead, J. S. Rennie, and N. Loveridge. 1993. In vivo effect of 1,25-dihydroxycholecalciferol on the proliferation and differentiation of avian chondrocytes. J. Bone Miner. Res. 8:1081–1088. Kashimura, J., M. Kimura, and Y. Itokawa. 1996. The effects of isomaltulose, isomalt, and isomaltulose-based oligomers on mineral absorption and retention. Biol. Trace Elem. Res. 54:239–250. Kim, W. K., L. M. Donalson, S. A. Bloomfield, H. A. Hogan, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2007. Molt performance and bone density of cortical, medullary, and cancellous bone in laying hens during feed restriction or alfalfa-based feed molt. Poult. Sci. 86:1821–1830. Kim, W. K., L. M. Donalson, A. D. Mitchell, L. F. Kubena, D. J. Nisbet, and S. C. Ricke. 2006. Effects of alfalfa and fructooligosaccharide on molting parameters and bone qualities using dual energy X-ray absorptiometry and conventional bone assays. Poult. Sci. 85:15–20. Kim, W. K., B. C. Ford, A. Mitchell, R. G. Elkin, and R. M. Leach Jr. 2004. Comparative assessment of bone of wild-type, restricted ovulator, and out of production hens. Br. Poult. Sci. 45:463–470. Kim, W. K., T. M. Herfel, C. S. Dunkley, P. Y. Hester, T. D. Crenshaw, and S. C. Ricke. 2008. The effects of alfalfa-based molt diets on skeletal integrity of white leghorns. Poult. Sci. 87:2178–2185. Kjaer, M. 2004. Role of extracellular matrix in adaptation of tendon and skeletal muscle to mechanical loading. Physiol. Rev. 84:649–698.
Key words: age, vitamin D, fructooligosaccharide, bone mineral density, mechanical property 2011 Poultry Science 90:2425–2432 doi:10.3382/ps.2011-01475
INTRODUCTION High growth rates in animals are generally associated with musculoskeletal weakness because of the development of bone that is much less organized, less dense, and more porous than the bones of slow-growing animals (Williams et al., 2004; Bennett, 2008). Rapid muscle growth in broiler chicks (meat-type chicks) through genetic selection causes imbalance between meat production and skeletal growth, resulting in abnormal skeletal development (Oviedo-Rondon et al., 2006). Leg problems and skeletal disorders, such as lameness, twisted legs, tibial dyschondroplasia (TD), and crooked toes, are the common causes of culling and mortality in broiler chicks (Bradshaw et al., 2002; Oviedo-Rondon ©2011 Poultry Science Association Inc. Received March 10, 2011. Accepted July 24, 2011. 1 Corresponding author: [email protected]
et al., 2006). Because leg problems in broilers cause pain, limit their mobility to access feed and water, and result in economic losses estimated at $120 million/yr (Cook, 2000), these are important animal welfare and economic issues (Oviedo-Rondon et al., 2009). One strategy that can be used to prevent leg problems and skeletal disorders is stimulating bone growth and strength in the early growth period. One potential nutrient or bioactive molecule is vitamin D3. Vitamin D3 is considered to be a prohormone generated in the skin through UV irradiation of 7-dehydrocholesterol or absorbed from the diet in the intestinal tract (DeLuca, 2004). It is not biologically active in this initial form and needs to be converted to 25-hydroxyvitamin D3 in the liver and subsequently to 1,25-dihydroxyvitamin D3 in the kidney (DeLuca, 2004). 1,25-Dihydroxyvitamin D3 acts through a nuclear receptor to express its numerous functions, including bone homeostasis, Ca, and phosphate absorption in the small intestine, Ca mobilization in bone, and Ca reabsorption in the kid-
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ABSTRACT A study was conducted to evaluate the effects of age, vitamin D3, and fructooligosaccharides (FOS) on bone mineral density (BMD), bone mineral content (BMC), cortical thickness, cortical and trabecular area, and mechanical properties in broiler chicks using peripheral quantitative computed tomography and mechanical testing. A total of 54 male broiler chicks (1 d old) were placed in battery brooders and fed a corn–soybean starter diet for 7 d. After 7 d, the chicks were randomly assigned to pens of 3 birds each. Each treatment was replicated 3 times. There were 6 treatments: 1) early age control (control 1); 2) control 2; 3) 125 µg/kg of vitamin D3; 4) 250 µg/kg of vitamin D3; 5) 2% FOS); and 6) 4% FOS. The control 1 chicks were fed a control broiler diet and killed on d 14 to collect femurs for bone analyses. The remaining groups were killed on d 21. Femurs from 3-wk-old chicks showed greater midshaft cortical BMD, BMC, bone area, thickness, and marrow area than those from 2-wk-old chicks
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bone growth and skeletal integrity in broiler chicks and that pQCT and mechanical testing would be excellent tools for evaluating bone quality and mechanical properties in broiler chicks. Therefore, the objective of this study was to evaluate the effects of age, vitamin D3, and FOS on BMD, BMC, cortical thickness, cortical area, marrow area, and mechanical properties in broiler chicks by pQCT and mechanical testing.
MATERIALS AND METHODS General Procedures A total of 54 male broiler chicks (1 d old) were placed in heated Petersime battery brooders (Petersime Incubator Co., Gettysburg, OH) and fed a corn–soybean broiler starter diet for 7 d. The starter diet contained 23% CP, 3,200 kcal/kg of ME, 1% Ca, 0.45% available P, and 90 µg/kg of vitamin D3. The chicks were individually weighed and then randomly assigned to pens of 3 birds each. Each treatment was replicated 3 times. There were 6 treatments: 1) early age control (control 1); 2) control 2; 3) 125 µg/kg of vitamin D3; 4) 250 µg/ kg of vitamin D3; 5) 2% FOS; and 6) 4% FOS. Vitamin D3 and FOS were purchased from Sigma-Aldrich (St. Louis, MO). The control 1 chicks were fed a control broiler diet and killed by cervical dislocation on d 14. The chicks from the other groups were fed a control broiler diet and treatment diets containing 125 µg/kg of vitamin D3, 250 µg/kg of vitamin D3, 2% FOS, or 4% FOS and were killed by cervical dislocation on d 21. For vitamin D3 treatments, 125 or 250 µg/kg of vitamin D3 was added to the control diet containing 90 µg/kg of vitamin D3. Left femurs were collected for BMD, BMC, bone area, bone thickness, and mechanical testing. The bones were cleaned of attached tissue, wrapped in PBS-soaked gauze for pQCT and mechanical testing, and stored at −20°C. All animal procedures were approved by the Texas A & M University Lab Animal Care Committee.
pQCT Bone scans of left femurs were performed with an XCT Research instrument (Stratec, Norland, Fort Atkinson, WI). This model has a minimum voxel size of 0.07 mm and a scanning beam thickness of 0.50 mm. Scan sites included the midshaft femurs (3 slices of each bone located at one-half the total bone length ± 2 mm) for volumetric BMD of the cortical bones; the proximal femurs were scanned to assess metaphyseal trabecular volumetric BMD (3 slices located 8.0, 8.5, and 9.0 mm from the proximal end of the bone). All scans were obtained at a scan speed of 2.5 mm/s, with a voxel resolution of 0.07 × 0.07 × 0.50 mm. In addition, the middiaphyseal cross-sectional moment of inertia (CSMI), which is a measure of bone material in a cross-section area and indicates its distribution around the neutral bending axis to resist bending (Bennett, 2008), was ob-
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ney (Farquharson et al., 1993; Leeson and Summers, 2001; DeLuca, 2004). It has been reported that the supplementation of high levels of vitamin D3 reduced incidence of leg problem, such as TD and Ca rickets in broilers (Whitehead et al., 2004; Atencio et al., 2005; Driver et al., 2006). However, few studies have used peripheral quantitative computed tomography (pQCT) and mechanical testing to evaluate whether high levels of vitamin D3 stimulate bone growth and enhance skeletal integrity in young broilers. Another potential bioactive molecule is fructooligosaccharide (FOS). Fructooligosaccharides are low molecular weight, nondigestible carbohydrates that have previously been examined as prebiotics in laying hens for their ability to limit Salmonella Enteritidis colonization and their influence on bone metabolism (Kim et al., 2006; Donalson et al., 2007, 2008a,b). Fructooligosaccharides also have the potential to increase bone formation and reduce bone resorption (Ohta et al., 1998; Takahara et al., 2000). Several studies have demonstrated that FOS enhanced true Ca, Mg, Fe, and P absorptions in the intestine of rats (Ohta et al., 1993, 1995; Kashimura et al., 1996; Morohashi et al., 1998). Moreover, FOS compounds have been shown to increase Ca balance and bone mineral density (BMD) in growing rats (Ohta et al., 1998). Takahara et al. (2000) observed that FOS increased femoral bone volume and mineral concentrations in rats. It has been shown that FOS induced intestinal Ca-binding protein (calbindin-D9k) levels in rats (Takasaki et al., 2000). More recently, Zafar et al. (2004) reported that FOS increased femoral Ca content and BMD whereas FOS reduced bone resorption rate in ovariectomized rats. However, no research has been conducted to evaluate the effects of FOS on bone growth and Ca metabolism in broiler chicks. Recently, dual-energy X-ray absorptiometry (DEXA) has been used to measure bone density and mineral content in broiler chicks and laying hens (Kim et al., 2004; Talaty et al., 2009). It has been demonstrated that BMD and bone mineral content (BMC) obtained by DEXA are positively correlated with bonebreaking force, bone ash, bone ash concentration, and bone weight in chickens (Mazzuco and Hester, 2005; Kim et al., 2006, 2008). Bone mineral density and BMC are highly correlated with each other and are good indicators for bone strength. However, few studies have evaluated bone quality in broiler chicks using pQCT, which can serve as a noninvasive method for measuring BMD, BMC, cross-section area, and 3-dimensional visualization of the bone (Clark et al., 2005). Because pQCT measures true volumetric bone density in specific sites (Korver et al., 2004; Kim et al., 2007), it can offer more precise and detailed information about site-specific BMD and cross-sectional geometry than DEXA. Thus, in the present study, we measured BMD and BMC in broiler chicks using pQCT. We hypothesized that high levels of vitamin D3 and FOS supplementation in broiler diets would increase
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tained with respect to the neutral bending axis of the femur bone shaft during 3-point bending (mechanical property testing).
Mechanical Testing
E = S × L3/(48,000 × CSMI), and
Effects of Age on Cortical BMD, BMC, Bone Area, Bone Thickness, and Mechanical Properties First, we evaluated the effects of age on bone growth and skeletal integrity of chick midshaft femurs (Table 1). Three-week-old chicks (control 2) had significantly higher BMD (P = 0.016) and BMC (P = 0.0003) compared with 2-wk-old chicks (control 1; Table 1). Femurs from 3-wk old chicks also showed greater midshaft cortical bone area (P = 0.0002), thickness (P = 0.01), and marrow area (P = 0.0001) than those from 2-wkold chicks. Next, we evaluated the effects of age on total, cortical, and trabecular BMD, BMC, and bone area of chick proximal femurs (Figure 1). Total, cortical, and trabecular BMD of chick proximal femurs were not influenced by age. However, BMC and bone area were significantly affected by age. Total, cortical, and trabecular BMC of control 2 chicks (3 wk old) were significantly higher than that of control 1 chicks (2-wk old; P = 0.0002, 0.04, and 0.003, respectively). Total and cortical bone areas and marrow area of control 2 chicks were also significantly larger than that of control 1 chicks (P = 0.0003, 0.01, and 0.0007, respectively). We further measured mechanical properties of chick femurs (Figure 2). The femurs of 2-wk-old chicks (control 1) exhibited significantly lower stiffness and ultimate load (structural properties) than those of 3-wkold chicks (control 2; Figure 2A and B; P = 0.0001), whereas ultimate stress and elastic modulus (material properties) of the femurs of 2-wk-old chicks were significantly higher than those of 3-wk-old chicks (Figure 2C and D; P = 0.0001). These results indicate that age is an important factor for bone growth and skeletal integrity in broiler chicks.
Effects of Vitamin D3 and FOS on BMD, BMC, Bone Area, Bone Thickness, and Mechanical Properties
US = UL × L × D/(8 × CSMI).
Statistical Analysis All data were subjected to 1-way ANOVA as a completely randomized design using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). Significant differences among the means were determined using Duncan’s multiple-range test at P < 0.05.
We evaluated the effects of vitamin D3 and FOS on bone growth and skeletal integrity in midshaft cortical bone of chick femurs (Figure 3). No significant differences were found in midshaft cortical BMD among the treatments (Figure 3A; P = 0.8306). However, chicks fed 250 µg/kg of vitamin D3 exhibited significantly
Table 1. Effects of broiler age on midshaft cortical bone mineral density (BMD), bone mineral content (BMC), bone area, bone thickness, and marrow area of femurs (n = 3/treatment) Treatment1
BMD (mg/cm3)
BMC (mg/mm)
Bone area (mm2)
Bone thickness (mm)
Marrow area (mm2)
Control 1 Control 2 Pooled SE P-value
692b 744a 8.5 0.016
9.2b 17.9a 0.51 0.0003
13.3b 24.1a 0.55 0.0002
1.33b 1.62a 0.04 0.01
8.02b 15.92a 0.17 0.0001
a,bMeans 1Control
within a column with different superscripts differ significantly (P < 0.05). 1 = 2-wk-old chicks; control 2 = 3-wk-old chicks.
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After pQCT scans were completed, mechanical properties of the midshaft femurs were determined by 3-point bending to failure with an Instron 1125 servo-controlled testing machine (Instron Corp., Canton, MA) according to previously published procedures (Allen and Bloomfield, 2003). The bones were thawed at room temperature and placed posterior side down on metal pin supports located ±10 mm (femurs) from the middiaphysis testing site. With a 455-kg load cell, quasistatic loading (2.5 mm/min) was applied to the anterior surface of the femurs until fractures occurred. All specimens were sprayed with PBS just before testing to maintain hydration. Displacements were monitored by a linear variable differential transformer interfaced with a personal computer. Raw data, collected at 10 Hz as load versus displacement curves, were analyzed with Table-Curve 2.0 software (Jandel, San Rafael, CA). Structural variables (ultimate load and stiffness) were obtained directly from the load:displacement curves. The maximum load obtained was defined as the ultimate load (UL, in N), and the slope of the elastic portion of the curve was defined as stiffness (S, in N/mm). Bone tissue material properties were calculated by normalizing structural properties for cross-sectional bone geometry at the site of testing by using CSMI (in mm4 from pQCT), bone diameter (D, in mm) as measured by calipers, and the appropriate bottom support span distance (L, which was 20 or 60 mm). The appropriate formulas for elastic modulus (E, in GPa) and ultimate stress (US, in MPa) were as follows:
RESULTS
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Figure 1. Effect of age on total, cortical, and trabecular bone mineral density (BMD), bone mineral content (BMC), and bone area of proximal femurs (n = 3/treatment). Control 1 = 2-wk-old chicks (early age control); control 2 = 3-wk-old chicks. Means with different letters differ significantly (P < 0.05).
higher midshaft cortical BMC than control 2, 2% FOS, or 4% FOS chicks (Figure 3B; P = 0.04). Furthermore, chicks fed 250 µg/kg of vitamin D3 had significantly larger midshaft cortical bone area and thickness compared with control 2, 2% FOS, or 4% FOS chicks (Figure 3C and D; P = 0.04 and 0.03, respectively). Bone marrow area of chicks fed 250 µg/kg of vitamin D3 was
significantly larger than that of control 2, 2% FOS, or 4% FOS chicks (Figure 3E; P = 0.01). In the present study, no significant differences were found in feed intake and weight gain among the treatment groups (data not shown; P = 0.643 and 0.478, respectively). We also determined total, cortical, trabecular BMD, BMC, and area and mechanical properties of proximal
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cal BMC than those fed 125 µg/kg of vitamin D3 or 2% FOS (Figure 5; P = 0.01). However, no significant differences were found in total, cortical, and bone marrow area among the treatments (Figure 6; P = 0.2676, 0.107, and 0.3325, respectively). We also evaluated the effects of vitamin D3 and FOS on mechanical properties. However, no significant differences were found in all mechanical properties (stiffness, ultimate load, ultimate stress, and elastic modulus) among the treatments (data not shown; P = 0.533, 0.949, 0.086, and 0.479, respectively).
DISCUSSION
femurs (Figures 4, 5, and 6). Total density of chicks fed 250 µg/kg of vitamin D3 was significantly higher than that of those fed 125 µg/kg of vitamin D3 or 2% FOS (Figure 4; P = 0.0487). However, no significant differences were found in cortical and trabecular BMD of proximal femurs among the treatments (Figure 4; P = 0.279 and 0.601, respectively). Chicks fed 250 µg/kg of vitamin D3 had significantly higher total and corti-
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Figure 2. Effect of age on A) stiffness, B) ultimate load, C) ultimate stress, and D) elastic modulus of chick femurs (n = 3/treatment). Control 1 = 2-wk-old chicks (early age control); control 2 = 3-wk-old chicks. Means with different letters differ significantly (P < 0.05).
In this study, we found that 3-wk-old chicks exhibited significantly greater midshaft cortical BMD, BMC, bone area, bone thickness, and marrow area than 2-wkold chicks, indicating that age is an important factor for bone growth and skeletal integrity. This result also indicates that endocortical resorption by osteoclasts (bone resorbing cells) occurs on the endosteal surface (inner edge of cortical shell) to increase marrow area while new bone formation by osteoblasts (bone forming cells) occurs on the periosteal surface (outside of cortical shell) to increase cortical bone area in broiler chicks as chick age increases. This is called radial or periosteal expansion (Bain and Watkins, 1993) and is a way to increase bone size and bone marrow area in broiler chicks as chick age increases. Interestingly, unlike midshaft femurs, total, cortical, and trabecular BMD values of proximal femurs were not influenced by age, whereas BMC and bone area of proximal femurs from 3-wk-old chicks were significantly higher than that of 2-wk-old chicks. These results suggest that bone density in specific sites of bones may be differently influenced by age. We also found that stiffness and ultimate load of femurs increased whereas ultimate stress and elastic modulus decreased as chick age increased. It appears that stiffness and ultimate load are negatively related to ultimate stress and elastic modulus in broiler chicks. Age-dependent changes of mechanical properties have been suggested by other researchers (Lawrence et al., 1994; Baumer et al., 2009). Our mechanical test showed that stiffness and ultimate load of chick femurs increased but ultimate stress and elastic modulus decreased as chick age increased. Because stiffness is important for effective locomotion (Kjaer, 2004), stiffer bones in older chicks might help improve the efficiency of leg movements. Furthermore, rapid growth in broiler chicks significantly reduces ultimate stress and elastic modulus independent of the bone size that confers increased stiffness and ultimate load. These results are in agreement with a swine study conducted by Baumer et al. (2009). Stiffness of the infant porcine parietal bone has been demonstrated to increase with age, whereas ultimate stress was reduced with age (Baumer et al., 2009). The results in the current study suggest that mechanical properties in broiler chicks are influenced by chick age.
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Figure 3. Midshaft cortical bone mineral density (BMD), bone mineral content (BMC), bone area, and bone thickness of femurs from 3-wk-old chicks (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides. Means with different letters differ significantly (P < 0.05).
In the present study, although no significant differences were found in midshaft cortical BMD among the treatments, chicks fed 250 µg/kg of vitamin D3 had significantly higher midshaft cortical BMC and larger bone area, bone thickness, and marrow area compared
with control. These results indicate that 250 µg/kg of vitamin D3 stimulates mineral deposition and radial growth of midshaft cortical bone in broilers. Increased rates of mineral deposition and radial growth by 250 µg/kg of vitamin D3 were well balanced in midshaft
BONE GROWTH AND SKELETAL INTEGRITY OF BROILER CHICKS
femurs to maintain similar BMD values to those of control. However, 250 µg/kg of vitamin D3 treatment did not change any mechanical properties. It also has been shown that chicks fed 250 µg/kg of vitamin D3 had significantly lower incidence of TD (Whitehead et al., 2004), a metabolic skeletal disorder in fast-growing meat-type poultry such as broilers and turkeys (Rath et al., 2004). Stimulating bone growth by high levels of vitamin D3 may be beneficial in reducing incidence of TD. However, FOS treatments did not affect any of the bone parameters. Although several studies have
Figure 6. Total, cortical, and marrow area of proximal femurs (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wkold chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/ kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides.
reported that FOS compounds increase BMD, BMC, and bone volume in rats (Ohta et al., 1998; Takahara et al., 2000; Zafar et al., 2004), FOS treatments did not increase BMC, BMD, bone area, or bone thickness in chicks. Species differential effects of FOS on bone and mineral metabolisms may exist. In summary, our study suggests that high levels of vitamin D3 can increase bone growth and mineral deposition in broiler chicks. However, FOS did not have any beneficial effects on bone growth and skeletal integrity. To reduce incidence of skeletal disorders, high levels of vitamin D3 dietary amendment may be a solution. Because chicks possess a high tolerance for vitamin D3, dietary supplementation of vitamin D3 higher than 250 µg/kg needs to be investigated to further confirm the potential for increases in bone growth and mineral contents, which in turn should lead to the reduction of skeletal problems in broiler chicks. Age is an important factor influencing skeletal integrity and mechanical properties in broiler chicks.
REFERENCES
Figure 5. Total, cortical, and trabecular bone mineral content (BMC) of proximal femurs (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides. Means with different letters differ significantly (P < 0.05).
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Figure 4. Total, cortical, and trabecular bone mineral density (BMD) of proximal femurs (n = 3/treatment). Control 2 = 3-wk-old chicks; 125 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 125 µg/kg of vitamin D3; 250 µg/kg Vit D3 = 3-wk-old chicks fed a broiler diet containing 250 µg/kg of vitamin D3; 2% FOS = 3-wk-old chicks fed a broiler diet containing 2% fructooligosaccharides; 4% FOS = 3-wk-old chicks fed a broiler diet containing 4% fructooligosaccharides. Means with different letters differ significantly (P < 0.05).
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