MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY Bone characteristics and femoral strength in commercial toms: The effect of protein and energy restriction R. C. Van Wyhe,* P. Regmi,* B. J. Powell,† R. C. Haut,† M. W. Orth,* and D. M. Karcher*1 *Department of Animal Sciences, and †Orthopaedic Biomechanics Laboratories, Michigan State University, East Lansing 48824
Key words: femur, turkey, break, ash, mechanics 2014 Poultry Science 93:943–952 http://dx.doi.org/10.3382/ps.2013-03604 pigs (Roberson et al., 2005; Shaw et al., 2006; Hertrampf et al., 2009). Thus, at a time when maximal support is needed, the bones are still trying to remodel to accommodate the weight, leaving it vulnerable to injury. Others have suggested similar issues with the increasing BW of skeletally immature broilers (Skinner and Waldroup, 1995). Growth rate of fast-growing meat birds has been reported to be mainly responsible for the increased porosity and reduced stiffness of bones (Williams et al., 2004). Thus, one way to reduce skeletal problems might be to restrict the growth of turkey toms and allow more time for the bones to mature to accommodate the BW. Feed-restricted 33- to 35-wk-old breeder tom femurs have been shown to have increased cross-sectional diameter, decreased cortical thickness, and decreased cross-sectional cortical area (Crespo et al., 2000). The smaller cortical thickness and cross-sectional area is likely a result of a smaller BW. However, when the bones were mechanically tested and data was adjusted for BW, the measurements translated into stiffer and stronger femurs in the feed-restricted turkeys (Crespo
INTRODUCTION The growth rate of the commercial turkey has approximately doubled over the last 45 yr (Havenstein et al., 2007). In toms, one problem likely related to increased size is the occurrence of femoral fractures. These fractures are a major concern to turkey producers, as the fractured femur may lacerate the femoral artery and result in death. Larger body mass in toms demands a concomitant increase in the size and strength of the skeletal structure for support. The bones respond to increased BW by continually increasing their rates of remodeling (Turner and Robling, 2004; Warden, 2006). The rates of systemic bone resorption in turkeys approaching market weight, as measured by serum pyridinoline (PYD) concentrations, are much higher than what are seen in other species with accelerated bone turnover, such as ovariectomized rats and market age ©2014 Poultry Science Association Inc. Received September 5, 2013. Accepted December 16, 2013. 1 Corresponding author: [email protected]
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mur length was longer in the 60% NRC birds at 5 and 10 kg of BW compared with control (P < 0.05); this significance was lost by the time birds reached 16 kg of BW. At 5 and 10 kg of BW, ash content was higher in the control birds than in the 60% NRC birds (P < 0.05). At 16 kg of BW, the 60% NRC birds had the highest femur ash (P < 0.05). The mechanical testing parameters were failure torque, shear strength, and shear modulus of the bones. The 60% diet produced the highest failure torque (P < 0.05), at 16 kg of BW and onward. The shear strength was greater (P = 0.01) once the birds reached 5 kg of BW for the 60% diet than other diets. In conclusion, reducing the energy and protein in the diet to 60% of NRC recommendations, thus slowing growth, improved bone strength, as measured by failure torque, and bone quality, as measured by shear strength, without altering bone length or ash content by the time birds reached market weight.
ABSTRACT Selection for rapid growth in turkeys has resulted in skeletal problems such as femoral fractures. Slowing growth rate has improved bone structure, but the effect on mechanical properties of the bone is unclear. The current study’s hypothesis was that slowing the growth of turkeys by reducing energy and CP in the diet would result in increased femur integrity. Commercial turkeys were fed 1 of 3 diets: control with 100% of NRC energy and CP levels, as well as a diet feeding 80 or 60% of NRC energy and CP levels. All other nutrients met or exceeded NRC requirements. Control birds were grown to 20 wk of age, whereas the 80 and 60% NRC birds were sampled when BW matched that of control birds at wk 4, 8, 12, 16, and 20. Both femurs were extracted, with one being measured and ashed and the other twisted to failure to evaluate mechanical properties. Total bone length, diameter, cortical thickness, and cortical density were measured. The total fe-
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VAN WYHE ET AL.
MATERIALS AND METHODS The experimental protocol was approved by the Michigan State University Institutional Animal Care and Use Committee.
Birds, Housing, and Diets The experiment consisted of 2 turkey strains (A and B) and 3 different diets with 4 replicate pens per diet and strain combination for a total of 600 toms. Three hundred one-day-old Hybrid toms and 300 one-day-old Nicholas toms were placed at day of hatch in an environmentally controlled house. All toms were started on a control starter for the first 7 d. On d 7, toms were weighed and reassigned to pens to ensure consistent average pen weights. Twenty-five poults were placed in each 8- × 10-ft pen with 12 pens of each strain. The industry control diet was formulated to meet or exceed the NRC (1994) recommendations. Experimental diets were formulated to limit amino acids and energy while maintaining other nutrients at or above NRC (1994) requirements. The 80% diet was formulated so that protein and energy met 80% of the recommend NRC (1994) value, whereas the 60% diet met 60% of the protein and energy requirement (Table 1). The 80 and 60% NRC diets contained extra fiber as a filler to obtain low protein and energy values. Diets were analyzed to confirm nutritive values (Table 2). Feed and water were provided ad libitum during the entire experi-
ment. Toms were raised to market weight (20 kg). Feed consumption and weight gain were recorded at each phase (every 4 wk) and average feed conversion ratio was calculated. Mortality was monitored continuously throughout the trial. Tom turkeys (80 and 60% NRC diets) were weight matched at the end of each phase (4, 8, 12, 16, and 20 wk; Table 3). At each sampling point, 2 average-weight toms per pen were selected for a total of 8 toms per diet and strain combination. The sampled birds had both femurs collected for analysis. The final collection of femur samples (n = 30 per treatment combination) occurred at slaughter, when toms achieved the 20-kg market weight.
PYD Brachial vein blood samples were collected and serum was separated and frozen at −20°C before analysis. Serum PYD concentration was quantified using commercially available ELISA tests (Quidel Corporation, San Diego, CA). Sample filtrate was diluted 8-fold in assay buffer for the analysis.
Peripheral Quantitative CT Imaging and Bone Ash Quantitative computed tomography scans of the left femurs were performed using a GE BrightSpeed scanner (General Electric Healthcare, Princeton, NJ). Total bone length was divided by 4 to obtain proximal (one-fourth), medial, and distal (three-fourths) regions. Mimics software (Materialise, Plymouth, MI) was used to analyze the resulting 1-mm cross-sections for diameter, cortical bone thickness, and cortical density in each of the 3 regions. Following tomographic scanning, the left femur was prepared for ashing. The femur was ether extracted, dried, and ashed according to methods described by Zhong et al. (2012).
Mechanical Testing The right femurs were allowed to thaw at 4°C for 10 h before preparation for mechanical tests. The bones were scraped with a scalpel at the proximal and distal ends to prevent slippage when potted in an air-hardening epoxy (Fiber Strand, Martin Senour Corp., Cleveland, OH). Each femur was then individually secured in a custom-built alignment fixture with a tri-axis bone clamp. The center of each bone was aligned vertically and horizontally with respect to the potting cups with the use of a laser-centering diode. The potting cups were separated by 1 of 3 spacers having lengths of 40.6, 60.9, and 81.7 mm (L1, L2, and L3, respectively). The spacers accommodated the increase in femur length with age. These distances were set based on an average length-to-diameter ratio of approximately 4 for the femurs in each age group. The 4-wk femurs were tested with L1, the 8-wk with L2, and the 12-, 16-, and 20-
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et al., 2000). It is unclear at what point these changes might start to take place and if a difference would be noted in commercial turkeys. Turner and Lilburn (1992) restricted growth in toms by feeding a control and a protein-deficient diet for the first 8 wk. The tibia and femur length was reduced in protein-deficient toms, but the effect was not observed after 12 to 14 wk. The width was, however, permanently reduced in both tibia and femur compared with the control group.. Whereas the bones were smaller, no difference in bone ash was observed between the 2 groups. It is unclear whether changes in size altered bone quality as the mechanical properties were not evaluated. In turkeys, most longitudinal bone growth has occurred by 10 wk of age (Lilburn et al., 2006), whereas mineralization has occurred at a fairly constant rate throughout the bird’s life, reaching a plateau as early as 5 wk (Applegate et al., 2006). With mineralization rates plateauing early in life, slowing the growth of the birds early may allow the skeletal system to better support BW and prevent femoral fractures that are normally seen as birds approach market weight. Therefore, the objective of the present study was to evaluate the structural and functional quality of appendicular bones in normal and feed-restricted turkeys by determining their mechanical properties, the alteration of strength and stiffness of the bone, and correlating rate of growth in birds to skeletal turnover.
0.32 0.27 0.05 1.42 3.08 0.51 0.05 0.04 — 0.25 2,850
—
43.83 48.00 — 2.19
Starter
0.22 0.15 0.01 1.47 2.58 0.46 0.05 — — 0.35 3,000
—
42.80 47.37 — 4.54
GP2-1
0.19 0.34 0.05 1.39 2.43 0.46 0.05 — — 0.35 3,150
—
55.61 34.13 — 5.00
GP-2
Control1
56.31 31.74 — 2.51 5.00 0.06 0.01 0.05 1.28 2.20 0.43 0.05 — — 0.35 3,270
GP-3 69.90 19.96 — 1.20 5.00 0.07 0.14 0.06 1.10 1.77 0.41 0.05 — — 0.35 3,325
Fin3 GP-1
GP-2
GP-3
Fin
17.89 32.11 35.00 45.00 40.00 37.00 31.37 29.80 20.00 13.00 39.26 25.00 25.00 25.00 30.00 0.54 1.00 1.80 0.91 0.72 — — — 1.00 5.00 0.11 0.13 0.01 0.02 — — 0.18 — 0.02 0.02 — 0.01 — 0.01 — 1.52 1.54 1.44 1.36 1.15 2.87 2.53 2.28 2.10 1.63 0.48 0.44 0.44 0.41 0.39 0.05 0.05 0.05 0.05 0.05 0.04 — — — — — 5.28 3.83 3.78 7.69 0.25 0.35 0.35 0.35 0.35 2,280 2,399 2,520 2,616 2,659
Starter
80%1 GP-1 20.92 22.24 25.00 1.00 — 0.09 0.14 0.01 1.52 2.67 0.45 0.05 — 25.55 0.35 1,800
Starter 8.35 26.03 40.00 0.60 — 0.07 — — 1.50 3.02 0.48 0.05 0.04 19.60 0.25 1,710
23.00 19.00 30.00 1.13 — 0.02 0.05 — 1.44 2.39 0.44 0.05 — 22.13 0.35 1,890
GP-2
60%1
30.00 11.61 30.00 0.44 1.00 0.02 0.06 0.03 1.35 2.19 0.42 0.05 — 22.48 0.35 1,961
GP-3
30.00 8.00 30.00 0.97 2.00 — 0.03 — 1.13 1.73 0.40 0.05 — 25.34 0.35 1,994
Fin
91.59 3,890 25.18 24.20 8.85 0.66 0.36 1.73 1.09 1.68 0.98 0.32 1.21
DM (%) Gross energy5 CP (%) Ca (%) Total P (%) Met (%) Cys (%) Lys (%) Ile (%) Arg (%) Thr (%) Trp (%) Phe (%)
90.44 4,133 26.56 17.29 7.22 0.56 0.38 1.70 1.15 1.74 0.97 0.34 1.26
GP2-1
91.05 4,060 21.55 16.37 6.47 0.49 0.32 1.62 0.93 1.43 0.85 0.26 1.05
GP-2
Control1
91.81 4,417 20.77 14.00 5.76 0.37 0.31 1.23 0.94 1.41 0.83 0.24 1.05
GP-3 91.16 4,260 14.15 13.12 5.15 0.30 0.23 0.95 0.67 0.98 0.61 0.22 0.74
Fin3 91.35 3,911 23.03 23.49 8.24 0.43 0.35 1.32 0.98 1.53 0.84 0.28 1.10
Starter 91.84 3,709 19.88 19.64 8.52 0.40 0.32 1.28 0.84 1.39 0.78 0.24 0.96
GP-1
91.42 3,831 19.50 16.27 7.83 0.31 0.31 1.14 0.84 1.35 0.75 0.25 0.96
GP-2
80%1
90.64 3,781 16.44 15.92 7.08 0.29 0.28 0.93 0.68 1.14 0.65 0.21 0.80
GP-3
91.87 3,687 11.64 12.62 5.03 0.20 0.22 0.69 0.52 0.81 0.48 0.16 0.62
Fin
GP-1 93.18 2,978 14.38 21.96 7.81 0.30 0.26 1.06 0.74 1.11 0.60 0.20 0.79
Starter 92.44 3,262 15.66 20.28 8.34 0.30 0.28 0.98 0.77 1.13 0.60 0.22 0.83
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93.34 3,059 14.87 15.06 9.25 0.24 0.25 0.95 0.68 1.12 0.59 0.21 0.75
GP-2
60%1
91.46 3,710 9.33 11.47 4.45 0.18 0.18 0.62 0.44 0.65 0.40 0.13 0.50
na4 na na na na na na na na na na na na
Fin
GP-3
1Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2GP = grower phase of the diet. 3Fin = finisher phase of the diet. 4na = missing data. 5kcal/kg.
Starter
Analyzed value
Table 2. Turkey diets analyzed nutrient values containing 100, 80, or 60% of NRC (1994) energy and protein requirements
1Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2GP = grower phase of the diet. 3Fin = finisher phase of the diet. 4kcal/kg.
Maize Soybean meal 48 Wheat middlings Soy oil White grease dl-Met Lys HCl Thr Limestone Dicalcium P Salt Copper sulfate Coban 90 Celite Vitamin-mineral premix Calculated ME4
Ingredient (%)
Table 1. Formulated turkey diets containing 100, 80, or 60% of NRC (1994) energy and protein requirements
FEED RESTRICTION ON TURKEY FEMURS
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VAN WYHE ET AL. Table 3. The sampling age for turkeys of equal BW fed a control or an energy- and protein-restricted diet Age (d) Treatment1 (kg of BW)
1
5
10
16
20
Control 80% 60%
28 28 35
56 56 78
84 88 111
112 117 155
140 140 178
1Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements.
τ=
2 ×Tf 2
(
πAB × 1 − γ 4
)
,
[1]
where a = the major inner radius; and b = the minor inner radius (as adapted from Young and Budynas, 2002). Bone quality before failure was quantified by computing its shear modulus using the expression G=
Tf × L K avg × ϕ
,
[3]
where L = the test length; φ = the failure angle of rotation; and Kavg = an average value of the structural torsion constant K, K =
(
πA3B 3 × 1 − γ 4 2
A +B
2
).
[4]
K was calculated using the average of the major and minor outer radii at the proximal, distal, and central locations. An average K was used to reduce error due to potential lengthwise variations along the femur (Levenston et al., 1994).
Statistical Analysis Performance parameters were analyzed using the PROC MIXED analysis of SAS (SAS Institute Inc., Cary, NC) with the LSMeans procedure. All comparisons between treatments were done on a BW basis. All bone measurements, such as length, section thickness, PYD, and ash data, were analyzed using the repeated measures statement. For repeated measurements, the model included the fixed effect of diet and pen, random effects of week, the interaction between diet and week, and the residual error. Differences between means were tested using the pdiff option of the lsmeans statement with significance accepted at P < 0.05. If a main effect of strain was observed, the 2 strains were analyzed independently.
RESULTS where Tf = the failure torque; A = the major outer radius; B = the minor outer radius; and 2γ =
a b + , A B
[2]
Performance The feed conversion ratio was higher for turkeys on the 60 and 80% NRC diets compared with control birds,
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wk with L3. Once the bone was in place, 4 set screws were threaded into each potting cup to accommodate fixation of the epoxy to the test fixture. Phosphatebuffered saline was routinely sprayed onto the exposed bone to prevent drying. After both ends of the specimen were potted, maximum and minimum diameter measurements were taken at proximal, distal, and central locations over the test length. Proximal and distal measurements were taken at the interface of the bone with the epoxy. The bone was then wrapped in PBSsoaked gauze and placed in a 4°C refrigerator for 24 h before testing. A servo-hydraulic testing machine (Model 1331, Instron Corp., Canton, OH) was adapted with a custom axial-to-torsion fixture to produce rotation control in the experiments. A torque load cell (Model 5330–1200, Interface Inc., Scottsdale, AZ) was attached to one rotation cup and used to capture force-time data. Angular rotation data were recorded with a rotary encoder fastened to a drive gear (Model BHW 16.05A72000BP-A, Baumer Electric Corp., Southington, CT). Experimental data was sampled at 1,000 Hz. Each specimen was secured into the fixture with screws into preexisting threads in the epoxy. The PBS gauzes were removed and the specimens were twisted to failure at a speed of 180°/sec. After fracture, the bone was removed from the fixture and 3 cortical thickness measurements were taken around the circumference of the fracture site with a digital caliper (IP66, Mitutoyo America). The structural strength of the bones was based on failure torque as measured by the peak torque generated in each experiment. The cross-sectional geometry of the femur was assumed to be a uniform thickness, hollow ellipse. To measure the structural quality of the bone as a material, its shear strength was calculated using
FEED RESTRICTION ON TURKEY FEMURS
947
with the largest cumulative feed conversion ratio (4.31) for the 60% group (P < 0.05; Figure 1). Feed intake was higher (P < 0.05) for birds on the 60% NRC diet compared with the 80% NRC diet and control, except for the period from 16 to 20 kg of BW. The 60% group took an additional 5 wk compared with the other treatments to reach the final BW of 20 kg (Table 1).
Average cortical thickness was largest in the 60% NRC group at 16 kg of BW, but smallest at 20 kg of BW (P < 0.05; Table 4). Strain A cortex was thickest at mid femur shaft for control birds at 16 kg of BW, whereas strain B was thickest in the 60% NRC group at 10, 16, and 20 kg of BW (P < 0.05; Table 5). The
Bone Physical Parameters and Quality Though the current study looked at factors that could contribute to bone weakness, no spiral fractures were recorded. The femur length increased with age and BW for each of the treatment groups. Birds fed the 60% diet had consistently longer femurs once the birds reached 5 kg of BW compared with control birds (P < 0.05; Figure 2). However, diameter along the proximal and distal femur shaft was widest in control birds at 20 kg and the 60% diet resulted in the narrowest distal diameter at the end of the trial (Table 4). A difference was noted between strains for the femoral diameter, cortical thickness, and cortical bone density only at mid diaphyseal shaft of the femur (Table 5). Strain A birds fed 80 and 60% NRC diets had a smaller medial diameter compared with the control group at 5 and again at 16 kg of BW. For strain B, control birds had widest medial diameter at 10 and 20 kg of BW (P < 0.05), whereas the 80% diet resulted in a similar diameter to the control and 60% group at 16 kg of BW.
Figure 2. The effect of dietary energy and protein restriction on femur length in commercial turkeys. Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements; significant differences (*) were defined as P < 0.05.
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Figure 1. The effect of dietary energy and protein restriction on feed intake and feed conversion of commercial toms. Means within same BW lacking a common superscript (a–c) differ significantly (P < 0.05). Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. FCR = feed conversion ratio.
948
VAN WYHE ET AL.
Table 4. Effect of dietary energy and protein restriction at a common BW on turkey proximal and distal femur parameters BW (kg) Item1
5
10
16
20
12.88 ± 0.39 12.60 ± 0.39 12.29 ± 0.39
20.60 ± 0.39 20.10 ± 0.39 21.05 ± 0.39
26.81 ± 0.39 27.00 ± 0.39 27.71 ± 0.39
30.51 ± 0.39 29.75 ± 0.39 29.56 ± 0.39
32.16 ± 0.26a 31.69 ± 0.26b 30.64 ± 0.26b
1.52 ± 0.07 1.45 ± 0.07 1.60 ± 0.07
1.71 ± 0.07 1.65 ± 0.07 1.70 ± 0.07
1.81 ± 0.07 1.71 ± 0.07 1.73 ± 0.07
764.57 ± 20.74b 842.06 ± 20.74a 872.83 ± 20.74a
1,175.96 ± 20.74 1,175.17 ± 20.74 1,182.78 ± 20.74
985.80 ± 20.74 983.98 ± 20.74 967.68 ± 20.74
11.67 ± 0.34 11.46 ± 0.34 11.79 ± 0.34
19.64 ± 0.34a 18.66 ± 0.34b 19.41 ± 0.34ab
25.48 ± 0.34 24.94 ± 0.34 25.54 ± 0.34
1.52 ± 0.09 1.52 ± 0.09 1.63 ± 0.09
2.02 ± 0.09a 1.93 ± 0.09ab 1.73 ± 0.09b
1.91 ± 0.09 1.73 ± 0.09 1.81 ± 0.09
865.47 ± 25.09b 988.66 ± 25.09a 924.54 ± 25.09ab
1,218.70 ± 25.09 1,219.60 ± 25.09 1,218.72 ± 25.09
1,098.19 ± 25.09ab 1,115.65 ± 25.09a 1,036.49 ± 25.09b
1.73 ± 0.07b 1.76 ± 0.07b 2.07 ± 0.07a 979.74 ± 20.74b 959.51 ± 20.74b 1,102.78 ± 20.74a 28.26 ± 0.34a 27.56 ± 0.34a 26.74 ± 0.34b 1.97 ± 0.09ab 1.89 ± 0.09b 2.19 ± 0.09a 1,129.83 ± 25.09ab 1,072.02 ± 25.09b 1,186.90 ± 25.09a
1.88 ± 0.04a 1.83 ± 0.04ab 1.72 ± 0.04b 997.91 ± 18.18b 985.92 ± 12.78b 1,032.44 ± 13.07a 29.55 ± 0.20a 28.84 ± 0.20b 27.13 ± 0.29c 1.74 ± 0.06b 1.89 ± 0.06ab 1.97 ± 0.08a 1,157.37 ± 14.46b 1,132.39 ± 14.83b 1,234.80 ± 22.36a
a,bMeans
within column for each treatment with no common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 1Control
distal cortical bone was thinnest in toms fed the 60% diet at 5 kg of BW but thickest at 20 kg of BW (P < 0.05; Table 4). The opposite trend was observed in the control birds during the same time period. Bone density analysis found that the birds fed the 60% diet had denser proximal and distal cortex after the birds were 16 kg (P < 0.05; Table 4). Table 5 indicates strain A birds’ medial femoral cortex was denser in the 80% group at 10 kg of BW but the control and 60% group had a more dense cortex at 16 kg of BW (P < 0.05). However, no difference in cortical density was observed between the treatment groups by the end of the trial. Strain A ash content was highest in the control group at 10 and 20 kg of BW, but lowest at 16 kg of BW (P < 0.05; Table 6). Strain B controls had more ash at 5 and 10 kg of BW, whereas the 60% group had the lowest (P < 0.05). However, at 16 kg of BW the percent ash flipped, with the control birds being lower than those fed the 60% diet (P < 0.05). Table 6 also reports tibia ash, but differences observed in strain B at 16 kg and strain A at 20 kg did not reflect differences observed in the femur ash.
PYD Serum PYD was measured as a marker of systemic bone resorption. Pyridinoline concentration increased with BW and age and peaked at 16 kg of BW for control birds, whereas it peaked at 10 kg of BW for the birds fed the 60 and 80% diets and then started de-
creasing until the end of the trial in strain A (Figure 3). As the birds reached 20 kg of BW, PYD was lowest in the 60% group regardless of strain. Strain B PYD levels peaked at 10 kg of BW in the 60% group, whereas birds fed the 80% diet and control toms peaked at 16 kg of BW (Figure 3). Strain B toms fed at the 60% diet had lower PYD levels at 16 and 20 kg of BW compared with control toms (P < 0.05; Figure 3).
Bone Strength Failure torque was not different between treatment groups until the birds reached 16 kg of BW (Table 7). At that point, the 60% group had the highest failure torque and the 80% group had the lowest (P < 0.05). Shear strength of all treatment groups increased from 5 to 20 kg of BW, with birds fed the 60% diet having the highest shear strength among the groups (P < 0.05). Shear modulus was greater in the 60% group compared with controls from 5 to 20 kg of BW, which was higher than birds fed the 80% diet at 10 and 16 kg of BW (P < 0.05).
DISCUSSION Previous research examined bone strength in breeder toms or young feed-restricted birds. The breeder toms in previous work were handled twice a week for semen collection. This could result in fractures or stresses to the legs of the turkey that are not seen in commercial
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Proximal diameter (mm) Control 80% 60% Proximal cortical thickness (mm) Control 80% 60% Proximal density (g/g) Control 80% 60% Distal diameter (mm) Control 80% 60% Distal cortical thickness (mm) Control 80% 60% Distal density (g/g) Control 80% 60%
1
1,778.8 ± 46.8 1,777.2 ± 46.8 1,849.1 ± 46.8
1,293.0 ± 46.8b 1,453.2 ± 46.8a 1,313.8 ± 46.8b
± 46.8 ± 46.8 ± 46.8
5
1,773.9 ± 47.8 1,756.1 ± 47.8 1,660.4 ± 47.8
2.33 ± 0.13 2.18 ± 0.13 2.11 ± 0.13
13.56 ± 0.32 12.91 ± 0.32 13.24 ± 0.32
B
2.58 ± 0.13a 2.04 ± 0.13b 2.32 ± 0.13ab
2.20 ± 0.13ab 2.02 ± 0.13b 2.49 ± 0.13a 1,638.4 ± 46.8a 1,571.3 ± 46.8b 1,774.4 ± 46.8a
19.94 ± 0.34a 18.62 ± 0.34b 18.16 ± 0.34b
A 20.37 ± 0.20a 19.43 ± 0.20b 19.03 ± 0.20b 2.46 ± 0.08 2.47 ± 0.08 2.48 ± 0.11
19.89 ± 0.32a 19.38 ± 0.32ab 18.71 ± 0.32b 1.98 ± 0.13b 2.21 ± 0.13ab 2.55 ± 0.13a
1,802.6 ± 26.9 1,761.2 ± 26.9 1,857.2 ± 41.5
A
B
1,686.7 ± 47.8 1,625.6 ± 47.8 1,632.9 ± 47.8
16
18.74 ± 0.32a 17.24 ± 0.32b 17.41 ± 0.32b
B
1,700.3 ± 46.8ab 1,679.4 ± 47.8 1,751.3 ± 46.8a 1,699.3 ± 47.8 1,576.9 ± 46.8b 1,599.5 ± 47.8
1.99 ± 0.13 1.98 ± 0.13 1.99 ± 0.13
17.44 ± 0.34 17.26 ± 0.34 16.47 ± 0.34
A
10
2.28 ± 0.13b 2.09 ± 0.13b 2.65 ± 0.13a
20.90 ± 0.21a 19.99 ± 0.20b 19.30 ± 0.02c
B
1,714.0 ± 29.1 1,698.7 ± 40.4 1,775.7 ± 27.7
20
45.40 ± 0.78 46.13 ± 0.78 45.07 ± 0.78
45.82 ± 0.77 45.53 ± 0.77 46.27 ± 0.77
± 0.66 ± 0.66 ± 0.66
B
± 0.84 ± 0.84 ± 0.84
A2 47.16 ± 0.78a 44.78 ± 0.78b 43.68 ± 0.78b 48.93 ± 0.77 49.19 ± 0.77 50.35 ± 0.77
50.24 ± 0.66a 47.92 ± 0.66b 50.49 ± 0.66a
B
50.01 ± 0.84 47.89 ± 0.84 48.55 ± 0.84
A
5
54.00 ± 0.66 53.57 ± 0.66 53.23 ± 0.66
49.97 ± 0.84a 47.63 ± 0.84b 45.56 ± 0.84b
A
10
53.84 ± 0.77 53.06 ± 0.77 52.43 ± 0.77
48.87 ± 0.78a 48.29 ± 0.78a 44.12 ± 0.78b
B
55.09 ± 0.66 55.76 ± 0.66 54.59 ± 0.66
48.90 ± 0.84b 48.97 ± 0.84b 52.61 ± 0.84a
A
16 A 51.27 ± 0.53a 51.51 ± 0.53a 48.99 ± 0.76b 54.00 ± 0.59b 55.91 ± 0.59a 55.61 ± 0.59a
B 44.67 ± 0.78b 46.44 ± 0.78b 50.07 ± 0.78a 53.55 ± 0.77a 50.56 ± 0.77b 53.21 ± 0.77a
1Control
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20
52.45 ± 0.77 52.92 ± 0.77 53.79 ± 0.77
49.12 ± 0.78 49.89 ± 0.78 49.76 ± 0.78
B
within column for each treatment with no common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2A = commercial genetic strain A; B = commercial genetic strain B.
a,bMeans
Femoral ash (%) Control 45.92 80% 45.06 60% 44.19 Tibial ash (%) Control 44.40 80% 43.88 60% 42.85
Item1
1
BW (kg)
Table 6. The effect of dietary energy and protein restriction on femoral and tibial ash in turkey toms
1Control
within column for each treatment with no common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2A = commercial genetic strain A; B = commercial genetic strain B.
a–cMeans
2.21 ± 0.13 1.94 ± 0.13 2.06 ± 0.13
1.63 ± 0.13 1.54 ± 0.13 1.54 ± 0.13
± 0.13 ± 0.13 ± 0.13
13.84 ± 0.34a 12.12 ± 0.34b 12.84 ± 0.34b
A
7.01 ± 0.32 6.83 ± 0.32 7.36 ± 0.32
B
± 0.34 ± 0.34 ± 0.34
A2
Diameter (cm) Control 7.25 80% 6.79 60% 6.98 Thickness (cm) Control 1.69 80% 1.39 60% 1.52 Density (g/g) Control 1,401.0 80% 1,403.2 60% 1,777.3
Item1
1
BW (kg)
Table 5. Effect of dietary energy and protein restriction on turkey on physical attributes of the medial portion of the femur of 2 different commercial strains
FEED RESTRICTION ON TURKEY FEMURS
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toms. Our work examines the difference in nutrient restriction in commercial toms until market weight. The performance result from the current study corroborates the findings of Turner and Lilburn (1992), in which they restricted protein in the toms’ diet for the first 8 wk and documented that the BW was lower in proteindeficient toms at 18 wk of age compared with normal toms. This suggests compensatory gain was insufficient to return protein-deficient toms to normal weight gain in the last 10 wk of growth. The length of the femur increased within birds of the same group with age and BW in the current study, which is in accordance with studies by Bond et al. (1991), Skinner and Waldroup
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Figure 3. The effect of dietary energy and protein restriction on the pyridinoline (PYD) in 2 commercial turkey strains. (A) The effect of dietary energy and protein restriction on the PYD of strain A. (B) The effect of dietary energy and protein restriction on the PYB of strain B. Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements; significant differences (*) were defined as P < 0.05.
(1995), and Bruno et al. (2007). Between the groups, nutrient-restricted birds had longer bones at the 10-kg mark and onward compared with control, which the age of the birds fed the 60% diet may account for. The difference in bone length may also be attributed to different early BW gains among the experimental groups with protein- and energy-restricted birds having lighter weights at the same point in life. Early mechanical loading in broiler chicks has been associated with accelerated cartilage resorption and early mineralization mediated by enhanced osteopontin and matrix metalloproteinases expression, resulting in narrower growth plates and shorter bones (Reich et al., 2005). Bruno et al. (2000) restricted growth at an earlier stage in broiler chickens and documented significant decreases in femur, tibia, and humerus lengths in the restricted group compared with the ad libitum group. Their results are contrary to the results of the current study, but the bone lengths in that study were averaged over the trial period and that does not take into account the compensatory growth that may have taken place later in the bird’s life. However, control birds had wider bone diameters, which might be an adaptive response to the compression forces occurring during a rapid weight gain (Akhter et al., 1998). Femurs of heavier commercial toms had wider cross-sectional areas than the slowgrowing strain of the same age, but differences observed were due to genotype, not diet (Zhong et al., 2012). When comparisons were made between the heavier and lighter birds of the same age and genetic strain, lighter birds had wider diameters of the proximal femur when data accounted for BW, while still maintaining the narrower cross-sectional area (Crespo et al., 2000). Crosssectional areas of the femur were not measured in our study. The cortical dimensions were altered by the nutrient restriction. Cortical thickness in protein- and energyrestricted birds, particularly the 60% group, caught up or even exceeded the control as the birds reached 20 kg of BW in all 3 regions of the bone, except for the proximal femur, indicating time is essential for bone to mature. A bone with thinner cortices is more prone to fracture (Crabtree et al., 2001), but the strength also depends upon the density of the bone. The wider cortical bone at the medial region, even comparing at common ages for 60% and control-fed birds, suggests that it might be the site where the femur experiences the major effect of mechanical loading and the subsequent bone formation taking place, making the bone stronger at this point. However, Cheng and Ward (2007) suggest no common skeletal orientation exists. Birds restricted in diet by 60% of nutritional requirements had denser femora in the proximal and distal regions of the bone compared with the other 2 groups after they reached 10 kg of BW. Toms fed the 60% diet had similar femoral density at 16 kg of BW compared with femoral density of 20-kg control toms. The results of cortical thickness and density indicate that maturation of cortical bone is more age-dependent rather than diet-dependent. A
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FEED RESTRICTION ON TURKEY FEMURS Table 7. The effect of dietary energy and protein restriction on femoral mechanical properties BW (kg) Item1 Failure torque (N·m) Control 80% 60% Shear strength (MPa) Control 80% 60% Shear modulus (GPa) Control 80% 60%
1
5
10
16
20
1.81 ± 0.94 1.95 ± 1.00 2.03 ± 1.00
10.67 ± 0.97 9.80 ± 0.94 11.72 ± 0.94
25.04 ± 0.97 23.52 ± 0.97 25.59 ± 0.94
38.43 ± 0.94b 33.44 ± 0.97c 40.73 ± 0.94a
48.44 ± 0.87b 41.98 ± 0.87c 52.06 ± 0.85a
42.76 ± 2.70b 53.74 ± 2.84a 44.56 ± 2.84b
30.10 ± 2.76b 33.92 ± 2.76b 37.65 ± 2.70a
36.38 ± 2.76b 40.57 ± 2.70b 53.62 ± 2.70a
58.31 ± 2.70b 55.26 ± 2.84b 70.99 ± 2.76a
70.54 ± 2.53b 72.30 ± 2.56b 79.62 ± 2.50a
1.57 ± 0.16b 1.64 ± 0.16b 2.10 ± 0.16a
2.07 ± 0.16b 2.02 ± 0.16b 2.27 ± 0.16a
0.86 ± 0.16b 1.16 ± 0.16a 1.00 ± 0.16ab
0.97 ± 0.16b 1.15 ± 0.16ab 1.31 ± 0.16a
2.30 ± 0.16b 2.36 ± 0.16ab 2.51 ± 0.16a
within same BW lacking a common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements.
comparison between the birds fed the 60% NRC diet at 10 kg (111 d of age) to control-fed birds at 16 kg (112 d of age) shows that the proximal cortical thickness and density, as well as the distal density, are not different at the same age, despite different BW (10 and 16 kg) for those birds. Turner and Lilburn (1992) found age-dependent rather than diet-dependent differences in bone ash in turkeys fed diets with restricted protein and Lys. Genetically slower growing toms had higher bone mineral content and better mechanical properties when their weight matched with commercial fastgrowing toms, suggesting matrix maturation and optimum mineralization takes time in meat turkeys (Zhong et al., 2012). A correlation of 93% between ash and whole BMD was reported for chickens (Onyango et al., 2003), but unlike the consistent improvement in density of proximal and distal femur in birds fed the 60% NRC diet, ash data were more variable at the end of the trial. A difference in age-related response of bone ash in the epiphyseal and diaphyseal region has been observed before (Turner and Lilburn, 1992). The variability observed in the present study could be age dependent rather than diet dependent. Also, ash measurement in the current study did not take into account the difference in the epiphyseal and diaphyseal section of femur and could be the reason for variable whole bone ash at the end of the trial. Shear strength and failure torque were higher for birds fed the 60% diet as they attained 10 kg of BW. This might be the result of an overall better density profile associated with this group. Increased bone mineral density is important for imparting stiffness to bones and will result in strong bones (Frost and Roland, 1991; Orban et al., 1993) together with appropriate collagen fiber orientation (Rath et al., 2000; Davison et al., 2006). The decreased load on the bone early in life may allow for the birds to reorient their bone structure to correspond to changing loads. In the trabecular bone of sheep, the bone adjusted and realigned itself in response to peak joint loading (Barak et al., 2011). Further studies into loading and mineral density
of turkey bones may help determine the mechanism besides the changes in bone strength. The serum PYD data showed a trend for a peak at around 10 kg of BW in the 60% nutrient-restricted groups of both strains and then started decreasing. In the control group, PYD peaked at 16 kg of BW and only started decreasing afterward. In birds from a previous study, serum PYD increased with age, peaking at wk 17 before decreasing (Roberson et al., 2005). Our results suggest that bone is experiencing a high turnover rate in the control and 80% group compared with the 60% group when market BW was achieved. This is probably due to the reduced nutrients in the diet rather than the differences in age or feed intake. Toms in the 60% group consumed more feed than control birds, but not more than the 80% group. The rate of systemic bone resorption observed in the current study suggests that even though fast-growing toms modulated the same geometry by increasing the diameter of femur, they could not attain optimal material strength, as mineralization and matrix maturation is time-dependent (Zhong et al., 2012). The results herein demonstrate that a manipulation of the growth rate via the diet during the production period affects numerous bone parameters. Bone mineral density and shear strength improved at the end of the trial when protein and energy was restricted by 60%, but some variations occurred throughout the study. Bone strength appears to correlate with bone density and also with stability of organic matrix, as shown by PYD. Though it is not economically advisable to increase feed conversion ratio and increase feed intake, a complete study with different restriction levels and their effect on bone quality could further elucidate the actual effects of nutrient restriction on bone quality in turkeys.
ACKNOWLEDGMENTS The authors thank the Animal Agriculture Initiative at Michigan State University for providing funding for
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a–cMeans
1Control
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VAN WYHE ET AL.
the project. We also thank Cara Robison for her assistance in sample collection and analysis, Angelo Napolitano, farm manager, and the rest of the farm crew at Michigan State University Poultry Research and Teaching Farm for their assistance in this study.
REFERENCES
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Akhter, M. P., D. M. Cullen, E. A. Pedersen, D. B. Kimmel, and R. R. Reeker. 1998. Bone response to in vivo mechanical loading in two breeds of mice. Calcif. Tissue Int. 63:442–449. Applegate, T. J., P. Jaynes, J. J. Dibner, M. Quiroz, and J. D. Richards. 2006. Growth and mineralization of the femur of the male turkey. Poult. Sci. 85(Suppl. 1):30. Barak, M. M., D. E. Lieberman, and J. J. Hublin. 2011. A Wolff in sheep’s clothing: Trabecular bone adaptation in response to changes in joint loading orientation. Bone 49:1141–1151. Bond, P. L., T. W. Sullivan, J. H. Douglas, and L. B. Robeson. 1991. Influence of age, sex, and method of rearing on tibia length and mineral deposition in broilers. Poult. Sci. 70:1936–1942. Bruno, L. D. G., R. L. Furlan, and E. B. Malheiros. 2000. Influence of early quantitative food restriction on long bone growth at different environmental temperatures in broiler chickens. Br. Poult. Sci. 41:389–394. Bruno, L. D. G., B. C. Luquetti, R. L. Furlan, and M. Macari. 2007. Influence of early qualitative feed restriction and environmental temperature on long bone development of broiler chickens. J. Therm. Biol. 32:349–354. Cheng, T., and T. Ward. 2007. Impact of Availa/M on growth, feed conversion, femur spiral fracture related mortality, bone morphometrics and carcass yield in tom turkeys. Poult. Sci. 86(Suppl. 1):781. Crabtree, N., N. Loveridge, M. Parker, N. Rushton, J. Power, and K. L. Bell. 2001. Intracapsular hip fracture and the region-specific loss of cortical bone: Analysis by peripheral quantitative computed tomography. J. Bone Miner. Res. 16:1318–1328. Crespo, R., S. M. Stover, K. T. Taylor, R. P. Chin, and H. L. Shivaprasad. 2000. Morphometric and mechanical properties of femora in young adult male turkeys with and without femoral fractures. Poult. Sci. 79:602–608. Davison, K. S., K. Siminoski, J. D. Adachi, D. A. Hanley, D. Goltzman, A. B. Hodsman, and R. Josse. 2006. Bone strength: The whole is greater than the sum of its parts. Semin. Arthritis Rheum. 36:22–31. Frost, T. J., and D. A. Roland. 1991. Current methods used in determination and evaluation of tibia strength: A correlation study involving birds fed various levels of cholecalciferol. Poult. Sci. 70:1640–1643. Havenstein, G. B., P. R. Ferket, J. L. Grimes, M. A. Qureshi, and K. E. Nestor. 2007. Comparison of the performance of 1966- versus 2003-type turkeys when fed representative 1966 and 2003 turkey diets: Growth rate, livability, and feed conversion. Poult. Sci. 86:232–240.
Hertrampf, T., B. Schleipen, C. Offermanns, M. Velders, U. Laudenbach, and P. Diel. 2009. Comparison of the bone protective effects of an isoflavone-rich diet with dietary and subcutaneous administrations of genistein in ovariectomized rats. Toxicol. Lett. 184:198–203. Levenston, M. E., G. S. Beaupre, and M. C. H. Van Der Meulen. 1994. Improved method for analysis of whole bone torsion tests. J. Bone Miner. Res. 9:1459–1465. Lilburn, M. S., A. Mitchell, and J. Anderson. 2006. The relationship between growth of commercial toms and linear skeletal development. Poult. Sci. 85(Suppl.1):31. NRC. 1994. Nutrient Requirements for Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Onyango, E. M., P. Y. Hester, R. L. Stroshine, and O. Adeola. 2003. Bone densitometry as an indicator of percentage tibia ash in broiler chicks fed varying dietary calcium and phosphorus levels. Poult. Sci. 82:1787–1791. Orban, J. L., D. A. Roland, and M. M. Bryant. 1993. Factors influencing bone mineral content, density, breaking strength and ash as a response to criteria for assessing bone quality in chickens. Poult. Sci. 72:437–446. Reich, A., N. Jaffe, A. Tong, I. Lavelin, O. Genina, M. Pines, D. Sklan, A. Nussinovitch, and E. Monsonego-Ornan. 2005. Weight loading young chicks inhibits bone elongation and promotes growth plate ossification and vascularization. J. Appl. Physiol. 98:2381–2389. Roberson, K. D., M. W. Klunzinger, R. A. Charbeneau, and M. W. Orth. 2005. Evaluation of phytase concentration needed for growing-finishing commercial turkey toms. Br. Poult. Sci. 46:470–477. Shaw, D. T., D. W. Rozeboom, G. M. Hill, M. W. Orth, D. S. Rosenstein, and J. E. Link. 2006. Impact of supplement withdrawal and wheat middling inclusion on bone metabolism, bone strength, and the incidence of bone fractures occurring at slaughter in pigs. J. Anim. Sci. 84:1138–1146. Skinner, J. T., and P. W. Waldroup. 1995. Allometric bone development in floor reared broilers. J. Appl. Poult. Res. 4:265–270. Turner, C. H., and A. G. Robling. 2004. Mechanical loading and bone formation. Bonekey. Osteovision 1:15–23. Turner, K. A., and M. S. Lilburn. 1992. The effect of early protein restriction (zero to eight weeks) on skeletal development in turkey toms from two to eighteen weeks. Poult. Sci. 71:1680–1686. Warden, S. J. 2006. Breaking the rule for bone adaptation to mechanical loading. J. Appl. Physiol. 100:1441–1442. 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. Young, W. C., and R. G. Budynas. 2002. Roark’s Formulas for Stress and Strain. 7th ed. McGraw-Hill, New York, NY. Zhong, Z., M. Muckley, S. Agcaoglu, M. E. Grisham, H. Zhao, M. Orth, M. S. Lilburn, O. Akkus, and D. M. Karcher. 2012. The morphological, material-level, and ash properties of turkey femurs from 3 different genetic strains during production. Poult. Sci. 91:2736–2746.
Key words: femur, turkey, break, ash, mechanics 2014 Poultry Science 93:943–952 http://dx.doi.org/10.3382/ps.2013-03604 pigs (Roberson et al., 2005; Shaw et al., 2006; Hertrampf et al., 2009). Thus, at a time when maximal support is needed, the bones are still trying to remodel to accommodate the weight, leaving it vulnerable to injury. Others have suggested similar issues with the increasing BW of skeletally immature broilers (Skinner and Waldroup, 1995). Growth rate of fast-growing meat birds has been reported to be mainly responsible for the increased porosity and reduced stiffness of bones (Williams et al., 2004). Thus, one way to reduce skeletal problems might be to restrict the growth of turkey toms and allow more time for the bones to mature to accommodate the BW. Feed-restricted 33- to 35-wk-old breeder tom femurs have been shown to have increased cross-sectional diameter, decreased cortical thickness, and decreased cross-sectional cortical area (Crespo et al., 2000). The smaller cortical thickness and cross-sectional area is likely a result of a smaller BW. However, when the bones were mechanically tested and data was adjusted for BW, the measurements translated into stiffer and stronger femurs in the feed-restricted turkeys (Crespo
INTRODUCTION The growth rate of the commercial turkey has approximately doubled over the last 45 yr (Havenstein et al., 2007). In toms, one problem likely related to increased size is the occurrence of femoral fractures. These fractures are a major concern to turkey producers, as the fractured femur may lacerate the femoral artery and result in death. Larger body mass in toms demands a concomitant increase in the size and strength of the skeletal structure for support. The bones respond to increased BW by continually increasing their rates of remodeling (Turner and Robling, 2004; Warden, 2006). The rates of systemic bone resorption in turkeys approaching market weight, as measured by serum pyridinoline (PYD) concentrations, are much higher than what are seen in other species with accelerated bone turnover, such as ovariectomized rats and market age ©2014 Poultry Science Association Inc. Received September 5, 2013. Accepted December 16, 2013. 1 Corresponding author: [email protected]
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mur length was longer in the 60% NRC birds at 5 and 10 kg of BW compared with control (P < 0.05); this significance was lost by the time birds reached 16 kg of BW. At 5 and 10 kg of BW, ash content was higher in the control birds than in the 60% NRC birds (P < 0.05). At 16 kg of BW, the 60% NRC birds had the highest femur ash (P < 0.05). The mechanical testing parameters were failure torque, shear strength, and shear modulus of the bones. The 60% diet produced the highest failure torque (P < 0.05), at 16 kg of BW and onward. The shear strength was greater (P = 0.01) once the birds reached 5 kg of BW for the 60% diet than other diets. In conclusion, reducing the energy and protein in the diet to 60% of NRC recommendations, thus slowing growth, improved bone strength, as measured by failure torque, and bone quality, as measured by shear strength, without altering bone length or ash content by the time birds reached market weight.
ABSTRACT Selection for rapid growth in turkeys has resulted in skeletal problems such as femoral fractures. Slowing growth rate has improved bone structure, but the effect on mechanical properties of the bone is unclear. The current study’s hypothesis was that slowing the growth of turkeys by reducing energy and CP in the diet would result in increased femur integrity. Commercial turkeys were fed 1 of 3 diets: control with 100% of NRC energy and CP levels, as well as a diet feeding 80 or 60% of NRC energy and CP levels. All other nutrients met or exceeded NRC requirements. Control birds were grown to 20 wk of age, whereas the 80 and 60% NRC birds were sampled when BW matched that of control birds at wk 4, 8, 12, 16, and 20. Both femurs were extracted, with one being measured and ashed and the other twisted to failure to evaluate mechanical properties. Total bone length, diameter, cortical thickness, and cortical density were measured. The total fe-
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MATERIALS AND METHODS The experimental protocol was approved by the Michigan State University Institutional Animal Care and Use Committee.
Birds, Housing, and Diets The experiment consisted of 2 turkey strains (A and B) and 3 different diets with 4 replicate pens per diet and strain combination for a total of 600 toms. Three hundred one-day-old Hybrid toms and 300 one-day-old Nicholas toms were placed at day of hatch in an environmentally controlled house. All toms were started on a control starter for the first 7 d. On d 7, toms were weighed and reassigned to pens to ensure consistent average pen weights. Twenty-five poults were placed in each 8- × 10-ft pen with 12 pens of each strain. The industry control diet was formulated to meet or exceed the NRC (1994) recommendations. Experimental diets were formulated to limit amino acids and energy while maintaining other nutrients at or above NRC (1994) requirements. The 80% diet was formulated so that protein and energy met 80% of the recommend NRC (1994) value, whereas the 60% diet met 60% of the protein and energy requirement (Table 1). The 80 and 60% NRC diets contained extra fiber as a filler to obtain low protein and energy values. Diets were analyzed to confirm nutritive values (Table 2). Feed and water were provided ad libitum during the entire experi-
ment. Toms were raised to market weight (20 kg). Feed consumption and weight gain were recorded at each phase (every 4 wk) and average feed conversion ratio was calculated. Mortality was monitored continuously throughout the trial. Tom turkeys (80 and 60% NRC diets) were weight matched at the end of each phase (4, 8, 12, 16, and 20 wk; Table 3). At each sampling point, 2 average-weight toms per pen were selected for a total of 8 toms per diet and strain combination. The sampled birds had both femurs collected for analysis. The final collection of femur samples (n = 30 per treatment combination) occurred at slaughter, when toms achieved the 20-kg market weight.
PYD Brachial vein blood samples were collected and serum was separated and frozen at −20°C before analysis. Serum PYD concentration was quantified using commercially available ELISA tests (Quidel Corporation, San Diego, CA). Sample filtrate was diluted 8-fold in assay buffer for the analysis.
Peripheral Quantitative CT Imaging and Bone Ash Quantitative computed tomography scans of the left femurs were performed using a GE BrightSpeed scanner (General Electric Healthcare, Princeton, NJ). Total bone length was divided by 4 to obtain proximal (one-fourth), medial, and distal (three-fourths) regions. Mimics software (Materialise, Plymouth, MI) was used to analyze the resulting 1-mm cross-sections for diameter, cortical bone thickness, and cortical density in each of the 3 regions. Following tomographic scanning, the left femur was prepared for ashing. The femur was ether extracted, dried, and ashed according to methods described by Zhong et al. (2012).
Mechanical Testing The right femurs were allowed to thaw at 4°C for 10 h before preparation for mechanical tests. The bones were scraped with a scalpel at the proximal and distal ends to prevent slippage when potted in an air-hardening epoxy (Fiber Strand, Martin Senour Corp., Cleveland, OH). Each femur was then individually secured in a custom-built alignment fixture with a tri-axis bone clamp. The center of each bone was aligned vertically and horizontally with respect to the potting cups with the use of a laser-centering diode. The potting cups were separated by 1 of 3 spacers having lengths of 40.6, 60.9, and 81.7 mm (L1, L2, and L3, respectively). The spacers accommodated the increase in femur length with age. These distances were set based on an average length-to-diameter ratio of approximately 4 for the femurs in each age group. The 4-wk femurs were tested with L1, the 8-wk with L2, and the 12-, 16-, and 20-
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et al., 2000). It is unclear at what point these changes might start to take place and if a difference would be noted in commercial turkeys. Turner and Lilburn (1992) restricted growth in toms by feeding a control and a protein-deficient diet for the first 8 wk. The tibia and femur length was reduced in protein-deficient toms, but the effect was not observed after 12 to 14 wk. The width was, however, permanently reduced in both tibia and femur compared with the control group.. Whereas the bones were smaller, no difference in bone ash was observed between the 2 groups. It is unclear whether changes in size altered bone quality as the mechanical properties were not evaluated. In turkeys, most longitudinal bone growth has occurred by 10 wk of age (Lilburn et al., 2006), whereas mineralization has occurred at a fairly constant rate throughout the bird’s life, reaching a plateau as early as 5 wk (Applegate et al., 2006). With mineralization rates plateauing early in life, slowing the growth of the birds early may allow the skeletal system to better support BW and prevent femoral fractures that are normally seen as birds approach market weight. Therefore, the objective of the present study was to evaluate the structural and functional quality of appendicular bones in normal and feed-restricted turkeys by determining their mechanical properties, the alteration of strength and stiffness of the bone, and correlating rate of growth in birds to skeletal turnover.
0.32 0.27 0.05 1.42 3.08 0.51 0.05 0.04 — 0.25 2,850
—
43.83 48.00 — 2.19
Starter
0.22 0.15 0.01 1.47 2.58 0.46 0.05 — — 0.35 3,000
—
42.80 47.37 — 4.54
GP2-1
0.19 0.34 0.05 1.39 2.43 0.46 0.05 — — 0.35 3,150
—
55.61 34.13 — 5.00
GP-2
Control1
56.31 31.74 — 2.51 5.00 0.06 0.01 0.05 1.28 2.20 0.43 0.05 — — 0.35 3,270
GP-3 69.90 19.96 — 1.20 5.00 0.07 0.14 0.06 1.10 1.77 0.41 0.05 — — 0.35 3,325
Fin3 GP-1
GP-2
GP-3
Fin
17.89 32.11 35.00 45.00 40.00 37.00 31.37 29.80 20.00 13.00 39.26 25.00 25.00 25.00 30.00 0.54 1.00 1.80 0.91 0.72 — — — 1.00 5.00 0.11 0.13 0.01 0.02 — — 0.18 — 0.02 0.02 — 0.01 — 0.01 — 1.52 1.54 1.44 1.36 1.15 2.87 2.53 2.28 2.10 1.63 0.48 0.44 0.44 0.41 0.39 0.05 0.05 0.05 0.05 0.05 0.04 — — — — — 5.28 3.83 3.78 7.69 0.25 0.35 0.35 0.35 0.35 2,280 2,399 2,520 2,616 2,659
Starter
80%1 GP-1 20.92 22.24 25.00 1.00 — 0.09 0.14 0.01 1.52 2.67 0.45 0.05 — 25.55 0.35 1,800
Starter 8.35 26.03 40.00 0.60 — 0.07 — — 1.50 3.02 0.48 0.05 0.04 19.60 0.25 1,710
23.00 19.00 30.00 1.13 — 0.02 0.05 — 1.44 2.39 0.44 0.05 — 22.13 0.35 1,890
GP-2
60%1
30.00 11.61 30.00 0.44 1.00 0.02 0.06 0.03 1.35 2.19 0.42 0.05 — 22.48 0.35 1,961
GP-3
30.00 8.00 30.00 0.97 2.00 — 0.03 — 1.13 1.73 0.40 0.05 — 25.34 0.35 1,994
Fin
91.59 3,890 25.18 24.20 8.85 0.66 0.36 1.73 1.09 1.68 0.98 0.32 1.21
DM (%) Gross energy5 CP (%) Ca (%) Total P (%) Met (%) Cys (%) Lys (%) Ile (%) Arg (%) Thr (%) Trp (%) Phe (%)
90.44 4,133 26.56 17.29 7.22 0.56 0.38 1.70 1.15 1.74 0.97 0.34 1.26
GP2-1
91.05 4,060 21.55 16.37 6.47 0.49 0.32 1.62 0.93 1.43 0.85 0.26 1.05
GP-2
Control1
91.81 4,417 20.77 14.00 5.76 0.37 0.31 1.23 0.94 1.41 0.83 0.24 1.05
GP-3 91.16 4,260 14.15 13.12 5.15 0.30 0.23 0.95 0.67 0.98 0.61 0.22 0.74
Fin3 91.35 3,911 23.03 23.49 8.24 0.43 0.35 1.32 0.98 1.53 0.84 0.28 1.10
Starter 91.84 3,709 19.88 19.64 8.52 0.40 0.32 1.28 0.84 1.39 0.78 0.24 0.96
GP-1
91.42 3,831 19.50 16.27 7.83 0.31 0.31 1.14 0.84 1.35 0.75 0.25 0.96
GP-2
80%1
90.64 3,781 16.44 15.92 7.08 0.29 0.28 0.93 0.68 1.14 0.65 0.21 0.80
GP-3
91.87 3,687 11.64 12.62 5.03 0.20 0.22 0.69 0.52 0.81 0.48 0.16 0.62
Fin
GP-1 93.18 2,978 14.38 21.96 7.81 0.30 0.26 1.06 0.74 1.11 0.60 0.20 0.79
Starter 92.44 3,262 15.66 20.28 8.34 0.30 0.28 0.98 0.77 1.13 0.60 0.22 0.83
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93.34 3,059 14.87 15.06 9.25 0.24 0.25 0.95 0.68 1.12 0.59 0.21 0.75
GP-2
60%1
91.46 3,710 9.33 11.47 4.45 0.18 0.18 0.62 0.44 0.65 0.40 0.13 0.50
na4 na na na na na na na na na na na na
Fin
GP-3
1Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2GP = grower phase of the diet. 3Fin = finisher phase of the diet. 4na = missing data. 5kcal/kg.
Starter
Analyzed value
Table 2. Turkey diets analyzed nutrient values containing 100, 80, or 60% of NRC (1994) energy and protein requirements
1Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2GP = grower phase of the diet. 3Fin = finisher phase of the diet. 4kcal/kg.
Maize Soybean meal 48 Wheat middlings Soy oil White grease dl-Met Lys HCl Thr Limestone Dicalcium P Salt Copper sulfate Coban 90 Celite Vitamin-mineral premix Calculated ME4
Ingredient (%)
Table 1. Formulated turkey diets containing 100, 80, or 60% of NRC (1994) energy and protein requirements
FEED RESTRICTION ON TURKEY FEMURS
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VAN WYHE ET AL. Table 3. The sampling age for turkeys of equal BW fed a control or an energy- and protein-restricted diet Age (d) Treatment1 (kg of BW)
1
5
10
16
20
Control 80% 60%
28 28 35
56 56 78
84 88 111
112 117 155
140 140 178
1Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements.
τ=
2 ×Tf 2
(
πAB × 1 − γ 4
)
,
[1]
where a = the major inner radius; and b = the minor inner radius (as adapted from Young and Budynas, 2002). Bone quality before failure was quantified by computing its shear modulus using the expression G=
Tf × L K avg × ϕ
,
[3]
where L = the test length; φ = the failure angle of rotation; and Kavg = an average value of the structural torsion constant K, K =
(
πA3B 3 × 1 − γ 4 2
A +B
2
).
[4]
K was calculated using the average of the major and minor outer radii at the proximal, distal, and central locations. An average K was used to reduce error due to potential lengthwise variations along the femur (Levenston et al., 1994).
Statistical Analysis Performance parameters were analyzed using the PROC MIXED analysis of SAS (SAS Institute Inc., Cary, NC) with the LSMeans procedure. All comparisons between treatments were done on a BW basis. All bone measurements, such as length, section thickness, PYD, and ash data, were analyzed using the repeated measures statement. For repeated measurements, the model included the fixed effect of diet and pen, random effects of week, the interaction between diet and week, and the residual error. Differences between means were tested using the pdiff option of the lsmeans statement with significance accepted at P < 0.05. If a main effect of strain was observed, the 2 strains were analyzed independently.
RESULTS where Tf = the failure torque; A = the major outer radius; B = the minor outer radius; and 2γ =
a b + , A B
[2]
Performance The feed conversion ratio was higher for turkeys on the 60 and 80% NRC diets compared with control birds,
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wk with L3. Once the bone was in place, 4 set screws were threaded into each potting cup to accommodate fixation of the epoxy to the test fixture. Phosphatebuffered saline was routinely sprayed onto the exposed bone to prevent drying. After both ends of the specimen were potted, maximum and minimum diameter measurements were taken at proximal, distal, and central locations over the test length. Proximal and distal measurements were taken at the interface of the bone with the epoxy. The bone was then wrapped in PBSsoaked gauze and placed in a 4°C refrigerator for 24 h before testing. A servo-hydraulic testing machine (Model 1331, Instron Corp., Canton, OH) was adapted with a custom axial-to-torsion fixture to produce rotation control in the experiments. A torque load cell (Model 5330–1200, Interface Inc., Scottsdale, AZ) was attached to one rotation cup and used to capture force-time data. Angular rotation data were recorded with a rotary encoder fastened to a drive gear (Model BHW 16.05A72000BP-A, Baumer Electric Corp., Southington, CT). Experimental data was sampled at 1,000 Hz. Each specimen was secured into the fixture with screws into preexisting threads in the epoxy. The PBS gauzes were removed and the specimens were twisted to failure at a speed of 180°/sec. After fracture, the bone was removed from the fixture and 3 cortical thickness measurements were taken around the circumference of the fracture site with a digital caliper (IP66, Mitutoyo America). The structural strength of the bones was based on failure torque as measured by the peak torque generated in each experiment. The cross-sectional geometry of the femur was assumed to be a uniform thickness, hollow ellipse. To measure the structural quality of the bone as a material, its shear strength was calculated using
FEED RESTRICTION ON TURKEY FEMURS
947
with the largest cumulative feed conversion ratio (4.31) for the 60% group (P < 0.05; Figure 1). Feed intake was higher (P < 0.05) for birds on the 60% NRC diet compared with the 80% NRC diet and control, except for the period from 16 to 20 kg of BW. The 60% group took an additional 5 wk compared with the other treatments to reach the final BW of 20 kg (Table 1).
Average cortical thickness was largest in the 60% NRC group at 16 kg of BW, but smallest at 20 kg of BW (P < 0.05; Table 4). Strain A cortex was thickest at mid femur shaft for control birds at 16 kg of BW, whereas strain B was thickest in the 60% NRC group at 10, 16, and 20 kg of BW (P < 0.05; Table 5). The
Bone Physical Parameters and Quality Though the current study looked at factors that could contribute to bone weakness, no spiral fractures were recorded. The femur length increased with age and BW for each of the treatment groups. Birds fed the 60% diet had consistently longer femurs once the birds reached 5 kg of BW compared with control birds (P < 0.05; Figure 2). However, diameter along the proximal and distal femur shaft was widest in control birds at 20 kg and the 60% diet resulted in the narrowest distal diameter at the end of the trial (Table 4). A difference was noted between strains for the femoral diameter, cortical thickness, and cortical bone density only at mid diaphyseal shaft of the femur (Table 5). Strain A birds fed 80 and 60% NRC diets had a smaller medial diameter compared with the control group at 5 and again at 16 kg of BW. For strain B, control birds had widest medial diameter at 10 and 20 kg of BW (P < 0.05), whereas the 80% diet resulted in a similar diameter to the control and 60% group at 16 kg of BW.
Figure 2. The effect of dietary energy and protein restriction on femur length in commercial turkeys. Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements; significant differences (*) were defined as P < 0.05.
Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 9, 2014
Figure 1. The effect of dietary energy and protein restriction on feed intake and feed conversion of commercial toms. Means within same BW lacking a common superscript (a–c) differ significantly (P < 0.05). Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. FCR = feed conversion ratio.
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VAN WYHE ET AL.
Table 4. Effect of dietary energy and protein restriction at a common BW on turkey proximal and distal femur parameters BW (kg) Item1
5
10
16
20
12.88 ± 0.39 12.60 ± 0.39 12.29 ± 0.39
20.60 ± 0.39 20.10 ± 0.39 21.05 ± 0.39
26.81 ± 0.39 27.00 ± 0.39 27.71 ± 0.39
30.51 ± 0.39 29.75 ± 0.39 29.56 ± 0.39
32.16 ± 0.26a 31.69 ± 0.26b 30.64 ± 0.26b
1.52 ± 0.07 1.45 ± 0.07 1.60 ± 0.07
1.71 ± 0.07 1.65 ± 0.07 1.70 ± 0.07
1.81 ± 0.07 1.71 ± 0.07 1.73 ± 0.07
764.57 ± 20.74b 842.06 ± 20.74a 872.83 ± 20.74a
1,175.96 ± 20.74 1,175.17 ± 20.74 1,182.78 ± 20.74
985.80 ± 20.74 983.98 ± 20.74 967.68 ± 20.74
11.67 ± 0.34 11.46 ± 0.34 11.79 ± 0.34
19.64 ± 0.34a 18.66 ± 0.34b 19.41 ± 0.34ab
25.48 ± 0.34 24.94 ± 0.34 25.54 ± 0.34
1.52 ± 0.09 1.52 ± 0.09 1.63 ± 0.09
2.02 ± 0.09a 1.93 ± 0.09ab 1.73 ± 0.09b
1.91 ± 0.09 1.73 ± 0.09 1.81 ± 0.09
865.47 ± 25.09b 988.66 ± 25.09a 924.54 ± 25.09ab
1,218.70 ± 25.09 1,219.60 ± 25.09 1,218.72 ± 25.09
1,098.19 ± 25.09ab 1,115.65 ± 25.09a 1,036.49 ± 25.09b
1.73 ± 0.07b 1.76 ± 0.07b 2.07 ± 0.07a 979.74 ± 20.74b 959.51 ± 20.74b 1,102.78 ± 20.74a 28.26 ± 0.34a 27.56 ± 0.34a 26.74 ± 0.34b 1.97 ± 0.09ab 1.89 ± 0.09b 2.19 ± 0.09a 1,129.83 ± 25.09ab 1,072.02 ± 25.09b 1,186.90 ± 25.09a
1.88 ± 0.04a 1.83 ± 0.04ab 1.72 ± 0.04b 997.91 ± 18.18b 985.92 ± 12.78b 1,032.44 ± 13.07a 29.55 ± 0.20a 28.84 ± 0.20b 27.13 ± 0.29c 1.74 ± 0.06b 1.89 ± 0.06ab 1.97 ± 0.08a 1,157.37 ± 14.46b 1,132.39 ± 14.83b 1,234.80 ± 22.36a
a,bMeans
within column for each treatment with no common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 1Control
distal cortical bone was thinnest in toms fed the 60% diet at 5 kg of BW but thickest at 20 kg of BW (P < 0.05; Table 4). The opposite trend was observed in the control birds during the same time period. Bone density analysis found that the birds fed the 60% diet had denser proximal and distal cortex after the birds were 16 kg (P < 0.05; Table 4). Table 5 indicates strain A birds’ medial femoral cortex was denser in the 80% group at 10 kg of BW but the control and 60% group had a more dense cortex at 16 kg of BW (P < 0.05). However, no difference in cortical density was observed between the treatment groups by the end of the trial. Strain A ash content was highest in the control group at 10 and 20 kg of BW, but lowest at 16 kg of BW (P < 0.05; Table 6). Strain B controls had more ash at 5 and 10 kg of BW, whereas the 60% group had the lowest (P < 0.05). However, at 16 kg of BW the percent ash flipped, with the control birds being lower than those fed the 60% diet (P < 0.05). Table 6 also reports tibia ash, but differences observed in strain B at 16 kg and strain A at 20 kg did not reflect differences observed in the femur ash.
PYD Serum PYD was measured as a marker of systemic bone resorption. Pyridinoline concentration increased with BW and age and peaked at 16 kg of BW for control birds, whereas it peaked at 10 kg of BW for the birds fed the 60 and 80% diets and then started de-
creasing until the end of the trial in strain A (Figure 3). As the birds reached 20 kg of BW, PYD was lowest in the 60% group regardless of strain. Strain B PYD levels peaked at 10 kg of BW in the 60% group, whereas birds fed the 80% diet and control toms peaked at 16 kg of BW (Figure 3). Strain B toms fed at the 60% diet had lower PYD levels at 16 and 20 kg of BW compared with control toms (P < 0.05; Figure 3).
Bone Strength Failure torque was not different between treatment groups until the birds reached 16 kg of BW (Table 7). At that point, the 60% group had the highest failure torque and the 80% group had the lowest (P < 0.05). Shear strength of all treatment groups increased from 5 to 20 kg of BW, with birds fed the 60% diet having the highest shear strength among the groups (P < 0.05). Shear modulus was greater in the 60% group compared with controls from 5 to 20 kg of BW, which was higher than birds fed the 80% diet at 10 and 16 kg of BW (P < 0.05).
DISCUSSION Previous research examined bone strength in breeder toms or young feed-restricted birds. The breeder toms in previous work were handled twice a week for semen collection. This could result in fractures or stresses to the legs of the turkey that are not seen in commercial
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Proximal diameter (mm) Control 80% 60% Proximal cortical thickness (mm) Control 80% 60% Proximal density (g/g) Control 80% 60% Distal diameter (mm) Control 80% 60% Distal cortical thickness (mm) Control 80% 60% Distal density (g/g) Control 80% 60%
1
1,778.8 ± 46.8 1,777.2 ± 46.8 1,849.1 ± 46.8
1,293.0 ± 46.8b 1,453.2 ± 46.8a 1,313.8 ± 46.8b
± 46.8 ± 46.8 ± 46.8
5
1,773.9 ± 47.8 1,756.1 ± 47.8 1,660.4 ± 47.8
2.33 ± 0.13 2.18 ± 0.13 2.11 ± 0.13
13.56 ± 0.32 12.91 ± 0.32 13.24 ± 0.32
B
2.58 ± 0.13a 2.04 ± 0.13b 2.32 ± 0.13ab
2.20 ± 0.13ab 2.02 ± 0.13b 2.49 ± 0.13a 1,638.4 ± 46.8a 1,571.3 ± 46.8b 1,774.4 ± 46.8a
19.94 ± 0.34a 18.62 ± 0.34b 18.16 ± 0.34b
A 20.37 ± 0.20a 19.43 ± 0.20b 19.03 ± 0.20b 2.46 ± 0.08 2.47 ± 0.08 2.48 ± 0.11
19.89 ± 0.32a 19.38 ± 0.32ab 18.71 ± 0.32b 1.98 ± 0.13b 2.21 ± 0.13ab 2.55 ± 0.13a
1,802.6 ± 26.9 1,761.2 ± 26.9 1,857.2 ± 41.5
A
B
1,686.7 ± 47.8 1,625.6 ± 47.8 1,632.9 ± 47.8
16
18.74 ± 0.32a 17.24 ± 0.32b 17.41 ± 0.32b
B
1,700.3 ± 46.8ab 1,679.4 ± 47.8 1,751.3 ± 46.8a 1,699.3 ± 47.8 1,576.9 ± 46.8b 1,599.5 ± 47.8
1.99 ± 0.13 1.98 ± 0.13 1.99 ± 0.13
17.44 ± 0.34 17.26 ± 0.34 16.47 ± 0.34
A
10
2.28 ± 0.13b 2.09 ± 0.13b 2.65 ± 0.13a
20.90 ± 0.21a 19.99 ± 0.20b 19.30 ± 0.02c
B
1,714.0 ± 29.1 1,698.7 ± 40.4 1,775.7 ± 27.7
20
45.40 ± 0.78 46.13 ± 0.78 45.07 ± 0.78
45.82 ± 0.77 45.53 ± 0.77 46.27 ± 0.77
± 0.66 ± 0.66 ± 0.66
B
± 0.84 ± 0.84 ± 0.84
A2 47.16 ± 0.78a 44.78 ± 0.78b 43.68 ± 0.78b 48.93 ± 0.77 49.19 ± 0.77 50.35 ± 0.77
50.24 ± 0.66a 47.92 ± 0.66b 50.49 ± 0.66a
B
50.01 ± 0.84 47.89 ± 0.84 48.55 ± 0.84
A
5
54.00 ± 0.66 53.57 ± 0.66 53.23 ± 0.66
49.97 ± 0.84a 47.63 ± 0.84b 45.56 ± 0.84b
A
10
53.84 ± 0.77 53.06 ± 0.77 52.43 ± 0.77
48.87 ± 0.78a 48.29 ± 0.78a 44.12 ± 0.78b
B
55.09 ± 0.66 55.76 ± 0.66 54.59 ± 0.66
48.90 ± 0.84b 48.97 ± 0.84b 52.61 ± 0.84a
A
16 A 51.27 ± 0.53a 51.51 ± 0.53a 48.99 ± 0.76b 54.00 ± 0.59b 55.91 ± 0.59a 55.61 ± 0.59a
B 44.67 ± 0.78b 46.44 ± 0.78b 50.07 ± 0.78a 53.55 ± 0.77a 50.56 ± 0.77b 53.21 ± 0.77a
1Control
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20
52.45 ± 0.77 52.92 ± 0.77 53.79 ± 0.77
49.12 ± 0.78 49.89 ± 0.78 49.76 ± 0.78
B
within column for each treatment with no common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2A = commercial genetic strain A; B = commercial genetic strain B.
a,bMeans
Femoral ash (%) Control 45.92 80% 45.06 60% 44.19 Tibial ash (%) Control 44.40 80% 43.88 60% 42.85
Item1
1
BW (kg)
Table 6. The effect of dietary energy and protein restriction on femoral and tibial ash in turkey toms
1Control
within column for each treatment with no common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements. 2A = commercial genetic strain A; B = commercial genetic strain B.
a–cMeans
2.21 ± 0.13 1.94 ± 0.13 2.06 ± 0.13
1.63 ± 0.13 1.54 ± 0.13 1.54 ± 0.13
± 0.13 ± 0.13 ± 0.13
13.84 ± 0.34a 12.12 ± 0.34b 12.84 ± 0.34b
A
7.01 ± 0.32 6.83 ± 0.32 7.36 ± 0.32
B
± 0.34 ± 0.34 ± 0.34
A2
Diameter (cm) Control 7.25 80% 6.79 60% 6.98 Thickness (cm) Control 1.69 80% 1.39 60% 1.52 Density (g/g) Control 1,401.0 80% 1,403.2 60% 1,777.3
Item1
1
BW (kg)
Table 5. Effect of dietary energy and protein restriction on turkey on physical attributes of the medial portion of the femur of 2 different commercial strains
FEED RESTRICTION ON TURKEY FEMURS
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VAN WYHE ET AL.
toms. Our work examines the difference in nutrient restriction in commercial toms until market weight. The performance result from the current study corroborates the findings of Turner and Lilburn (1992), in which they restricted protein in the toms’ diet for the first 8 wk and documented that the BW was lower in proteindeficient toms at 18 wk of age compared with normal toms. This suggests compensatory gain was insufficient to return protein-deficient toms to normal weight gain in the last 10 wk of growth. The length of the femur increased within birds of the same group with age and BW in the current study, which is in accordance with studies by Bond et al. (1991), Skinner and Waldroup
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Figure 3. The effect of dietary energy and protein restriction on the pyridinoline (PYD) in 2 commercial turkey strains. (A) The effect of dietary energy and protein restriction on the PYD of strain A. (B) The effect of dietary energy and protein restriction on the PYB of strain B. Control = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements; significant differences (*) were defined as P < 0.05.
(1995), and Bruno et al. (2007). Between the groups, nutrient-restricted birds had longer bones at the 10-kg mark and onward compared with control, which the age of the birds fed the 60% diet may account for. The difference in bone length may also be attributed to different early BW gains among the experimental groups with protein- and energy-restricted birds having lighter weights at the same point in life. Early mechanical loading in broiler chicks has been associated with accelerated cartilage resorption and early mineralization mediated by enhanced osteopontin and matrix metalloproteinases expression, resulting in narrower growth plates and shorter bones (Reich et al., 2005). Bruno et al. (2000) restricted growth at an earlier stage in broiler chickens and documented significant decreases in femur, tibia, and humerus lengths in the restricted group compared with the ad libitum group. Their results are contrary to the results of the current study, but the bone lengths in that study were averaged over the trial period and that does not take into account the compensatory growth that may have taken place later in the bird’s life. However, control birds had wider bone diameters, which might be an adaptive response to the compression forces occurring during a rapid weight gain (Akhter et al., 1998). Femurs of heavier commercial toms had wider cross-sectional areas than the slowgrowing strain of the same age, but differences observed were due to genotype, not diet (Zhong et al., 2012). When comparisons were made between the heavier and lighter birds of the same age and genetic strain, lighter birds had wider diameters of the proximal femur when data accounted for BW, while still maintaining the narrower cross-sectional area (Crespo et al., 2000). Crosssectional areas of the femur were not measured in our study. The cortical dimensions were altered by the nutrient restriction. Cortical thickness in protein- and energyrestricted birds, particularly the 60% group, caught up or even exceeded the control as the birds reached 20 kg of BW in all 3 regions of the bone, except for the proximal femur, indicating time is essential for bone to mature. A bone with thinner cortices is more prone to fracture (Crabtree et al., 2001), but the strength also depends upon the density of the bone. The wider cortical bone at the medial region, even comparing at common ages for 60% and control-fed birds, suggests that it might be the site where the femur experiences the major effect of mechanical loading and the subsequent bone formation taking place, making the bone stronger at this point. However, Cheng and Ward (2007) suggest no common skeletal orientation exists. Birds restricted in diet by 60% of nutritional requirements had denser femora in the proximal and distal regions of the bone compared with the other 2 groups after they reached 10 kg of BW. Toms fed the 60% diet had similar femoral density at 16 kg of BW compared with femoral density of 20-kg control toms. The results of cortical thickness and density indicate that maturation of cortical bone is more age-dependent rather than diet-dependent. A
951
FEED RESTRICTION ON TURKEY FEMURS Table 7. The effect of dietary energy and protein restriction on femoral mechanical properties BW (kg) Item1 Failure torque (N·m) Control 80% 60% Shear strength (MPa) Control 80% 60% Shear modulus (GPa) Control 80% 60%
1
5
10
16
20
1.81 ± 0.94 1.95 ± 1.00 2.03 ± 1.00
10.67 ± 0.97 9.80 ± 0.94 11.72 ± 0.94
25.04 ± 0.97 23.52 ± 0.97 25.59 ± 0.94
38.43 ± 0.94b 33.44 ± 0.97c 40.73 ± 0.94a
48.44 ± 0.87b 41.98 ± 0.87c 52.06 ± 0.85a
42.76 ± 2.70b 53.74 ± 2.84a 44.56 ± 2.84b
30.10 ± 2.76b 33.92 ± 2.76b 37.65 ± 2.70a
36.38 ± 2.76b 40.57 ± 2.70b 53.62 ± 2.70a
58.31 ± 2.70b 55.26 ± 2.84b 70.99 ± 2.76a
70.54 ± 2.53b 72.30 ± 2.56b 79.62 ± 2.50a
1.57 ± 0.16b 1.64 ± 0.16b 2.10 ± 0.16a
2.07 ± 0.16b 2.02 ± 0.16b 2.27 ± 0.16a
0.86 ± 0.16b 1.16 ± 0.16a 1.00 ± 0.16ab
0.97 ± 0.16b 1.15 ± 0.16ab 1.31 ± 0.16a
2.30 ± 0.16b 2.36 ± 0.16ab 2.51 ± 0.16a
within same BW lacking a common superscript differ significantly (P < 0.05). = birds fed a diet containing 100% of NRC (1994) protein and energy requirements; 80% = birds fed a diet containing 80% of NRC (1994) protein and energy requirements; 60% = birds fed a diet containing 60% of NRC (1994) protein and energy requirements.
comparison between the birds fed the 60% NRC diet at 10 kg (111 d of age) to control-fed birds at 16 kg (112 d of age) shows that the proximal cortical thickness and density, as well as the distal density, are not different at the same age, despite different BW (10 and 16 kg) for those birds. Turner and Lilburn (1992) found age-dependent rather than diet-dependent differences in bone ash in turkeys fed diets with restricted protein and Lys. Genetically slower growing toms had higher bone mineral content and better mechanical properties when their weight matched with commercial fastgrowing toms, suggesting matrix maturation and optimum mineralization takes time in meat turkeys (Zhong et al., 2012). A correlation of 93% between ash and whole BMD was reported for chickens (Onyango et al., 2003), but unlike the consistent improvement in density of proximal and distal femur in birds fed the 60% NRC diet, ash data were more variable at the end of the trial. A difference in age-related response of bone ash in the epiphyseal and diaphyseal region has been observed before (Turner and Lilburn, 1992). The variability observed in the present study could be age dependent rather than diet dependent. Also, ash measurement in the current study did not take into account the difference in the epiphyseal and diaphyseal section of femur and could be the reason for variable whole bone ash at the end of the trial. Shear strength and failure torque were higher for birds fed the 60% diet as they attained 10 kg of BW. This might be the result of an overall better density profile associated with this group. Increased bone mineral density is important for imparting stiffness to bones and will result in strong bones (Frost and Roland, 1991; Orban et al., 1993) together with appropriate collagen fiber orientation (Rath et al., 2000; Davison et al., 2006). The decreased load on the bone early in life may allow for the birds to reorient their bone structure to correspond to changing loads. In the trabecular bone of sheep, the bone adjusted and realigned itself in response to peak joint loading (Barak et al., 2011). Further studies into loading and mineral density
of turkey bones may help determine the mechanism besides the changes in bone strength. The serum PYD data showed a trend for a peak at around 10 kg of BW in the 60% nutrient-restricted groups of both strains and then started decreasing. In the control group, PYD peaked at 16 kg of BW and only started decreasing afterward. In birds from a previous study, serum PYD increased with age, peaking at wk 17 before decreasing (Roberson et al., 2005). Our results suggest that bone is experiencing a high turnover rate in the control and 80% group compared with the 60% group when market BW was achieved. This is probably due to the reduced nutrients in the diet rather than the differences in age or feed intake. Toms in the 60% group consumed more feed than control birds, but not more than the 80% group. The rate of systemic bone resorption observed in the current study suggests that even though fast-growing toms modulated the same geometry by increasing the diameter of femur, they could not attain optimal material strength, as mineralization and matrix maturation is time-dependent (Zhong et al., 2012). The results herein demonstrate that a manipulation of the growth rate via the diet during the production period affects numerous bone parameters. Bone mineral density and shear strength improved at the end of the trial when protein and energy was restricted by 60%, but some variations occurred throughout the study. Bone strength appears to correlate with bone density and also with stability of organic matrix, as shown by PYD. Though it is not economically advisable to increase feed conversion ratio and increase feed intake, a complete study with different restriction levels and their effect on bone quality could further elucidate the actual effects of nutrient restriction on bone quality in turkeys.
ACKNOWLEDGMENTS The authors thank the Animal Agriculture Initiative at Michigan State University for providing funding for
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the project. We also thank Cara Robison for her assistance in sample collection and analysis, Angelo Napolitano, farm manager, and the rest of the farm crew at Michigan State University Poultry Research and Teaching Farm for their assistance in this study.
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