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METABOLISM AND NUTRITION: Research Notes |
* Department of Nutrition, Dietetics and Food Science, and Department of Statistics, Brigham Young University, Provo, Utah 84602
2 Corresponding author: susan_fullmer{at}byu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.01). Bone mineral content determined by ashing was significantly different by 9.2% (P 0.0001) from BMC determined in vivo by DXA; however, there were no differences in ex vivo BMC and BMC ash, although they were highly correlated (r = 0.99, P 0.0001). We concluded that there was no effect of n-3 fatty acids on tibia bone in mature White Leghorn chickens. The GE Lunar Prodigy DXA instrument significantly underestimated the in vivo BMC in chickens.
Key Words: chicken • n-3 fatty acid • bone strength • bone mineral density • dual-energy x-ray absorptiometry validation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Essential fatty acids may also modulate bone strength. Liu et al. (2003) demonstrated that long-term supplementation of n-3 fatty acids was beneficial to biomechanical properties. In quail fed fish oil, compared with chicken fat and soybean oil, bone shear force and shear stress were increased. Reinwald et al. (2004) studied n-3-deficient rats, which had significantly lower peak force compared with n-3-adequate controls. Load at failure and bending moment were also significantly lower in n-3-deficient rats. These results were found in tibia bones, yet the same mechanical tests performed on the corresponding femur bones did not differ significantly between n-3-deficient rats and n-3-adequate rats. This seems to indicate that different bones respond to diets differently. Further, these studies may be limited by the fact that they generally reported only 1 parameter of bone strength (peak force or failure point, for example), but a true picture of the biomechanical properties of bone is represented by several biomechanical parameters.
Few animal studies have addressed the effects of n-6 and n-3 fatty acids on bone strength characteristics. Most studies have been conducted in quail and rats. In one study in White Leghorns, Mazzuco et al. (2005) found little effect of pre- and postmolt diets on n-6:n-3 ratios.
Therefore, the specific aims of this research were to evaluate the effect of feeding diets of varying ratios of n-6:n-3 fatty acids for 42 wk on 1) bone morphology (weight, length, diameter), 2) BMD and BMC, and 3) 7 parameters of bone breaking strength in mature laying chickens. We hypothesized that BMC, BMD, and bone breaking strength would be improved as the n-6:n-3 ratio diminished.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Birds received the diets for 42 wk. The experimental diets were isonitrogenous and isocaloric and were formulated to meet or exceed the nutrient requirements of laying chickens (NRC, 1994). Birds were cared for following the Guidelines for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Poultry Science Association, 1999). The birds were exposed to a lighting regimen of 14L:10D and a constant ambient temperature of 18°C. Experimental rations and water were provided ad libitum.
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BMD and BMC
A dual-energy x-ray absorptiometry (DXA) instrument (GE Lunar Prodigy, GE Healthcare, Madison, WI) was used to measure BMD (g/cm2) and BMC (g). All chickens in the study were scanned in the last month before sacrifice to obtain in vivo BMD and BMC. Chickens were strapped supine onto a Plexiglas plate with Velcro restraints at the neck, breast, and legs. Wings were bound in front. The left tibia bone was isolated to scan, with the leg and body oriented consistently throughout the scans. Birds were scanned twice at each measurement while positioned on the Plexiglas. The mean of the 2 scans was recorded.
Before breaking, excised left tibia bones were scanned by DXA to obtain ex vivo BMD and ex vivo BMC to compare against ash content. Ex vivo tibias were submerged in a container of white rice and were scanned in the same anatomical orientation.
Bone Morphology
At 58 wk of age, chickens were killed by CO2 gassing. The left tibia bones were excised from each chicken. Bones were freed of the surrounding soft tissue and cartilage and weighed in grams. Bone length (from the longitudinal epicondyle to the distal end) and diameter at the middiaphysis were measured with 6-in. (15.2 cm) dial calipers in centimeters. After measurements were obtained, the left tibias were wrapped in saline-soaked towels, sealed in plastic bags, and frozen (–80°C) until the day of scanning and determination of breaking strength and ashing. All samples were kept on ice at the time of collection and then frozen at –80°C.
Bone Mechanical Testing
A 3-point bending test was used to determine the mechanical properties of the left tibia bone. Specimens were thawed at room temperature in plastic bags and kept moist during testing. Force in compression was applied by a 3-point bending rig using a 50-kg loading cell (TA.XT2 Texture Analyzer, Texture Technology Corp., Scarsdale, NY). While loaded at the midpoint of the shaft, with fulcrum points 62.33 mm apart, the tibia was subjected to shear test to failure at a constant loading rate of 0.5 mm/s. Fulcrum points and speeds were based on recommendations from the American Society of Agricultural Engineers (2001) shear and 3-point bending test of animal bone (standard S459). The load vs. deformation curve was read from the texture analyzer. From this curve, the ultimate force required to break a bone was recorded in newtons.
With the calculations given below (Turner and Burr, 1993; American Society of Agricultural Engineers, 2001), load or force was converted to stress, and deformation was converted to strain to create a stress-strain curve. From this curve, slope, area under the curve, bending stress, and maximum strain were determined:
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where c is the distance from the cross-sectional center of the mass [(diameter height/2) x 0.57559]; d is the distance the beam traveled; F is the force; L is the length between supports; and I is the moment of inertia, 0.0549 x[(B xD3) –(b xd3)], where B is the outside major diameter (mm), b is the inside major diameter (mm), D is the outside minor diameter (mm), and d is the inside minor diameter (mm).
The slope of the elastic region of the stress-strain curve (Young’s modulus of elasticity) is considered a measure of the intrinsic stiffness or rigidity of a material. Using linear regression, we determined the slope of the stress-strain curve between 25 and 75% of ultimate failure. The area under the stress-strain curve (N/mm3), a measure of toughness or the amount of energy needed to cause a fracture, was calculated by using SAS 9.1 (SAS Institute, 2003).
An important distinction in mechanical properties is the difference between extrinsic and intrinsic properties. Extrinsic properties refer to the whole bone or bone specimen, and thus reflect the combined effects of bone size and shape in addition to tissue material properties. Intrinsic properties refer to only the tissue-level material behavior, which allows bone size and shape to be factored out. In this study, all strength variables calculated are intrinsic measurements, with the exception of ultimate force.
Cortical Thickness
Following bone breaking, the inner and outer diameters of tibia bones were measured with a micrometer. Cortical thickness was derived by the formula [(D –d) + (B –d)]/ 4 (American Society of Agricultural Engineers, 2001).
Ash Content
Tibia samples were oven-dried (Thelco Laboratory Oven, Thermo Electron Corporation, Pittsburgh, PA) at 101°C for 24 h. They were weighed to obtain a dry weight and ashed in a muffle furnace (Series 550 Isotemp Muffle Furnace, Fisher Scientific, Pittsburgh, PA) at 500°C for 24 h in porcelain crucibles. Remaining ash was weighed to determine total tibial ash BMC.
Statistical Analysis
Data were analyzed by using GLM ANOVA (SAS version 9.1, SAS Institute, 2003). Significant differences of means between treatments were tested by using the Tukey-Kramer post hoc test at the 5% probability level. Variation within each treatment was expressed as the SEM. Pearson correlations were performed among selected variables of interest.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Egg Fatty Acid Profile
As intended, 18:3 
-linolenic acid and 22:6 docosahexaenoic acid (DHA) increased incrementally as the dietary ratio of n-6 to n-3 decreased. Total n-3 fatty acids were lowest in the highest n-6 group (47.8:1) and were highest in the highest n-3 diet group (4.7:1). The egg fatty acid analysis verified that diets were fed correctly and that dietary lipids were reflected in the fatty acid profile of the eggs.
BMD and BMC
There was no significant effect of diet on BMD (either in vivo or ex vivo) or BMC (in vivo, ex vivo, or ash) at the end of study.
Bone Morphology and Bone Strength Parameters
There was no significant difference among diet groups for bone morphology measurements (weight, length, or outer diameter; data not shown); however, there were significant differences in inner diameter, resulting in significant differences in cortical thicknesses (see Table 2). Strength characteristics are reported in Table 2. There were no other significant effects of diet on measures of bone strength.
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0.0001). Bone mineral content in vivo was significantly different from BMC ex vivo (2.76 ± 0.057 g vs. 3.10 ± 0.057 g, respectively; Tukey-Kramer posthoc adjusted P 0.0002). Bone mineral content in vivo was also significantly different from BMC ash (2.76 ± 0.057 g vs. 3.04 ± 0.057 g, adjusted P 0.0025), a difference of 9.2%. However, BMC ex vivo and BMC ash were not significantly different (BMC ex vivo 3.10 ± 0.057 g, BMC ash 3.04 ± 0.057 g) but were highly correlated (r = 0.99, P 0.0001). The Pearson correlation between BMC in vivo and ex vivo was r = 0.89 (P 0.0001). The Pearson correlation between BMC in vivo and BMC ash was r = 0.90 (P 0.0001). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was no significant difference among dietary groups in final in vivo and ex vivo BMD or BMC as measured by DXA. A study by Johnston et al. (2006) demonstrated similar findings in turkeys. The BMD and BMC of turkey breeder hens were not significantly different between 2 extreme diet treatments, one rich in n-3 fatty acids (n-6:n-3 ratio of 1.7:1) and the other high in n-6 fatty acids (n-6:n-3 ratio of 19:1). Mazzuco et al. (2005) fed an n-6:n-3 ratio of 0.6 or 8.0 in the diets of chickens and found no effect on the decline in BMD between the 2 diets. A rat study by Mollard et al. (2005) resulted in no significant difference in bone area, BMD, or BMC between diet groups fed diets high in n-3 vs. diets high in n-6.
However, other animal studies have shown a positive effect of n-3 on BMD and BMC. Liu et al. (2003) found a significantly higher BMC in quail fed a fish oil-supplemented diet (high in n-3) compared with a soybean oil diet group (high in n-6). Similarly, fish oil-supplemented rats had significantly higher BMD in the distal femur and proximal tibia than a corn oil-supplemented group (high n-6; Sun et al., 2003; Bhattacharya et al., 2005). A probable explanation for the dichotomy in animal studies concerning the effects of n-6:n-3 fatty acids on BMD and BMC could be the age of the animals. Most avian and rat n-6:n-3 studies have been initiated close to birth. Our study looked at the effect of various dietary ratios of n-6:n-3 fatty acids on mature (58-wk-old) White Leghorn chickens. The laying chicken is still growing until approximately 32 wk of age. Liu et al. (2004) investigated the effects of dietary lipids on mature quail BMC at 8 mo of age and found similar results as in other research on young animals, yet the dietary treatments were initiated at 1 mo of age. This allowed the quail to be fed the diets from early on in growth to maturity. In contrast, our study began dietary treatments at 4 mo of age until chickens were 15 mo of age. It is possible that dietary lipids did not have as pronounced an effect on mature bone as on growing, forming bone. Although bone is constantly being resorbed and renewed, dietary ratios of n-6:n-3 may not modulate this process in mature bone.
We compared ex vivo and ash BMC to in vivo BMC of the tibia to determine the precision and accuracy of the GE Lunar Prodigy DXA instrument in unanesthetized live birds. The percentage CV, a measure of precision, of the GE Lunar Prodigy DXA instrument for measuring in vivo BMC was 2%. Accuracy, or how accurately the Prodigy measured the true BMC compared with bone ash BMC as the gold standard), was 9.2%. The GE Lunar Prodigy instrument significantly underestimated in vivo BMC. In contrast, Schreiweis et al. (2005) found that the Norland DXA instrument overestimated in vivo BMC in live chickens. We feel that accuracy within 10% in a longitudinal repeated-measures study is adequate as long as precision is acceptable.
Consistent with the findings of no effect of n-3 fatty acids on bone characteristics, dietary ratios of n-6 to n-3 did not influence any bone strength parameters except cortical thickness. Liu et al. (2003) showed that high n-3 fatty acids increased the strength of bone in quail. Quail fed soybean oil diets (high in n-6) had significantly lower values in shear force and stress than those fed fish oil or hydrogenated soybean oil. Reinwald et al. (2004) also found that n-3 fatty acid-deficient rats had significantly lower peak force compared with n-3-adequate controls. Load at failure and bending moment were also significantly lower in n-3-deficient rats. These results were found in tibia bones, yet the same mechanical tests performed on the corresponding femur bones did not differ significantly between n-3-deficient rats and n-3-adequate rats. This may indicate that different bones respond to diets differently.
An interesting finding was a significant increase in cortical thickness as n-3 fatty acids increased until the highest n-3 diet was given. Cortical thickness in the first 4 diets demonstrated a stepwise increase until the highest n-3 diet (4.7:1), and then dropped sharply to the level of the lowest n-3 diet (47.8:1) and was significantly lower than in the 5.9:1 and 7.6:1 diets. This is possibly a quadratic response; however, one would not expect such a dramatic change between the 5.9:1 and 4.7:1 diets. If the response were truly quadratic, one might expect an earlier plateau and not such a dramatic difference between minimally different dietary ratios. Yet despite a greater cortical thickness, it did not result in stronger bone. Unfortunately, we did not examine the microarchitecture to determine whether there was an impact on the microstructure of bone.
Our results of no effect of n-3 on bone are contrary to a number of studies (Watkins et al., 1996, 2000; Weiler and Fitzpatrick-Wong, 2002; Liu et al., 2003, 2004; Weiss et al., 2005). The dietary source of n-3 fatty acids may be a significant factor. Flax oil was the primary source of n-3 fatty acids via -linolenic acid in this study, whereas fish oil was the primary source of n-3 fatty acids in similar animal studies (Watkins et al., 1996, 1997; Judex et al., 2000; Liu and Denbow, 2001; Sun et al., 2003; Liu et al., 2004; Bhattacharya et al., 2005; Mollard et al., 2005). Fish oil is a good source of DHA and eicosapentaenoic acid (EPA), as opposed to feeding -linolenic acid, which has a low to minimal conversion rate to DHA and EPA. It is unknown whether the potential effects of n-3 fatty acids on bone are due to specific longer chain derivatives, such as DHA and EPA. Thus, the differences in specific n-3 sources on bone have yet to be clarified.
This study has several limitations. We analyzed only the tibia bone, and it may not reflect the characteristics of other bones. Further, we did not analyze the microstructure of bone or the organic collagen matrices.
In conclusion, the present investigation demonstrated that various ratios of n-6:n-3 in the diets of mature White Leghorn chickens did not affect bone characteristics or bone strength parameters except cortical thickness, which was significantly increased as n-3 in the diet increased until the highest n-3 diet. However, an increase in cortical thickness did not ultimately affect bone strength. Thus, further research should focus on determining whether n-3 fatty acids are more effective in developing bones. Research should also determine the potential effect of cortical thickness on bone strength.
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FOOTNOTES |
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Received for publication May 9, 2007. Accepted for publication October 13, 2007.
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REFERENCES |
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