Poult Sci 2006. 85:1648-1651
© 2006 Poultry Science Association
PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION |
Use of Apparent Transverse Quantitative Ultrasonography to Assess Skeletal Integrity in Layers
M. A. Martinez-Cummer*,
M. Hurtig
and
S. Leeson*
* Department of Animal and Poultry Science, and Department of Clinical Studies, University of Guelph, Ontario, Canada N1G 2W1
1 Corresponding author: sleeson{at}uoguelph.ca
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ABSTRACT
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Although there are several techniques currently available to assess skeletal integrity in live birds, few offer important features for application in a commercial setting, offering ease of use and moderate cost. Quantitative ultrasonography (QUS) is an established technique for diagnosis of osteoporosis in humans and horses that has potential application for layers. An OmniSense 700S quantitative ultrasonometer was evaluated for use with Single Comb White Leghorn hens. Humeral QUS values (m/s) were measured in a series of experiments using a total of 144 Shaver White hens. Significant correlations (P < 0.01) were observed among sequential QUS measurements taken on the same bird at 54, 60, and 66 wk of age. At the completion of the studies (66 wk of age), the left and right humeri were excised, cleaned, and rescanned. Postmortem QUS data from left and right humeri were related (R2 = 0.72, P < 0.0001), although future studies may need to consider both sides of the skeleton to account for asymmetry conditions. Ultrasound data collected from live hens at 66 wk of age correlated well with postmortem QUS data (R2 = 0.80, P < 0.0001). Quantitative ultrasonography did not correlate with humeral bone-breaking force measured postmortem. Bones from live hens, surrounded by tissue thicker than 4 mm, could not be read by the QUS probe.
Key Words: quantitative ultrasonography • osteoporosis • laying hen
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INTRODUCTION
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In commercial layers, optimal skeletal integrity is vital to ensure provision of some of the Ca needed for eggshell formation and Ca homeostasis during acute or chronic Ca deficiency. The former may be caused by a lack of a supply of Ca at night, when the hen does not eat and when most eggshell formation takes place (Etches, 1987). However, factors such as logistical errors in feed mills and farms, lower feed intake during hot summer months, competition for access to feed, and decreased intestinal absorption as hens age (Elaroussi et al., 1994) are examples of factors that contribute to a long-term or transient Ca imbalance. Selection for increased egg production seen in modern layer strains means that pullets reach sexual maturity at a young age and rely more on their skeletal Ca reserves to maintain the Ca demand for eggshell synthesis. Whitehead and Fleming (2000) indicated that this potentially leads to a problem in bone remodeling and, as a consequence, leads to a higher incidence of osteoporosis in older commercial flocks. Currently, many processing plants are refusing to process older laying hens because of the high incidence of bone splinters, a situation that leads to loss of revenue for egg producers and associated costs for alternate methods of disposal (Whitehead and Wilson, 1992).
Traditional methodology to assess skeletal integrity in poultry has usually required destructive tests (Rowland et al., 1967; Harner and Wilson, 1986; Newman and Leeson, 1998). Quantitative ultrasonography (QUS) is an established diagnostic technique in human medicine for bone assessment involving clinical osteoporosis. Quantitative ultrasonography is particularly interesting to the poultry industry because it does not involve ionizing radiation, is cost-effective, and is easily transportable. The technique relies on correlating properties of bone with time of propagation of sound waves. Quantitative ultrasonography was used to measure the speed of sound of the humeral midshaft on the caudal surface in live hens. This anatomical site was found to consistently produce a signal with reproducible results. On the other hand, measurements in the femur, tibia, and tarsometatarsus were found to be inconsistent because of surrounding tissue interference. A physical limitation with this technique is that signal emissions from currently available QUS probes are only capable of penetrating up to 4 mm.
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MATERIALS AND METHODS
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In Vivo Evaluation of Skeletal Integrity Using QUS.
Preliminary tests were conducted on live birds and, subsequently, on humeral specimens from hens of various ages and BW groups in an attempt to identify suitable anatomical sites. An anatomical site was identified in both the distal and proximal ends of the humerus in live hens before scanning. By palpating live birds on the caudal side of the left humerus, it was determined that the incisura capitis was consistently identified at the proximal region and the processus flexorius at the distal region, and these were used as reference locations.
Birds were selected from studies that involved nutritional programs aimed at producing a range of skeletal development. All diets were composed of corn and soybean meal and met requirements (NRC, 1994) for all nutrients other than Ca. Calcium levels from 18 to 24 wk were 2.5, 3.5, or 4.5%. After these times, birds were fed diets meeting NRC (1994) requirements and generally in line with commercial standards. Samples were prepared to test humeral speed of sound using a Sunlight Omnisense 7000S ultrasound bone densitometer (Sunlight Medical Ltd., Rehovot, Israel) on the caudal surface of the left humeri in live hens at 54, 60, and 66 wk of age. The incisura capitis and the processus flexorius were identified with a fine-point marker. Subsequently, vernier calipers with a precision of ±0.05 mm were used to measure the length of the humerus based on these anatomical sites, and a line was drawn using a fine-point marker; this line was then used to estimate the midpoint of the humerus. The midpoint of the probe was then positioned over the midpoint of the humerus for measurement of QUS. Ultrasonic gel was used as a coupling medium during the measurement (Figure 1
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Ex Vivo Evaluation of Skeletal Integrity Using QUS.
For postmortem humeral evaluation, the same hens used for in vivo measurements were transferred to the University of Guelph processing plant and were stunned by electrical shock and subsequently killed by exsanguination. Both humeri were removed, defleshed, the length from the distal to the proximal end of the bone specimens was measured using vernier calipers (±0.05 mm), and the line was drawn on the bone at midshaft using a fine-point marker. The midpoint of the caudal surface probe was aligned with the midpoint of the humerus and rescanned with the QUS probe.
Humeral Breaking Strength Evaluated with the 3-Point Bending Test.
The 3-point bending test was used to test the breaking strength of the left humeri from 48 hens at 66 wk of age. The specimens were prepared by removing excess flesh and then drying at room temperature (22°C) for 7 d. The length from the distal and proximal end of the bone was measured using vernier calipers, and a line was drawn on the bone at midshaft. The bending test was conducted using an Instron universal material testing machine fitted with a 1-kN load cell (model 4204, Instron Corp., Canton, MA). The crosshead movement was set at a rate of 5.0 mm/min for all tests. Bones were placed dorsal side on rounded supports 7.0 cm apart and were aligned so that the descending probe would make contact with the bone at the midshaft point. The value for the ultimate breaking strength or force was obtained by loading the bone until the ultimate failure point of the structure was reached and reported in newtons.
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RESULTS AND DISCUSSION
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Fleming et al. (2004) recently reported the use of a bone ultrasound unit designed to measure speed of sound through the phalanges of the human hand. The unit was used for monitoring the distal end of the first phalanx of the third toe in laying hens and is in agreement with data obtained with our speed-of-sound data. However, these authors reported 1,850 ± 5.1 m/s for caged hens; this value is less than half the apparent humeral ultrasound velocity found in our control-fed hens at 3,891 ± 50.16 m/s. These differences appear to be associated with the types of bone evaluated. The ultrasonic waves emitted by the unit used by Fleming et al. (2004) apparently traveled through cortical and substantial amounts of cancellous bone across the third proximal phalanx, whereas the unit used in our studies was expected to measure mostly humeral cortical bone. These data suggest ultrasonic velocity depends on the type of bone that the sound waves are traveling through, and so bone descriptions are essential for comparison of data from different studies.
Correlations were calculated among QUS measurements made on the same birds at different ages to determine if early measurements (54 wk of age) were accurate predictors of later measurements (Table 1). Moderate to high correlations were found among data taken over time. Thus, earlier measurements of humeral speed of sound may be useful predictors of humeral speed of sound toward the end of the laying cycle (66 wk of age) and seem repeatable for individual birds; therefore, they may be predictive of any future skeletal deterioration.
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Table 1. Correlation values for in vivo apparent transverse speed of sound (m/s) of the humerus of Single Comb White Leghorn hens at 54 vs. 60 vs. 66 wk of age, as determined by quantitative ultrasonography (m/s) in 3 independent nutrition studies
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Although the correlations in the present studies between left and right humeri were generally high (R2 = 0.72, P < 0.0001), the variation observed in the relationship between left and right humeri (Figure 2) suggest that future evaluations may need to consider both sides of the skeleton in the analysis to account for an asymmetry in skeletal integrity that potentially could introduce a source of variation. This may be particularly critical in commercial conditions where a series of potentially critical variables (that are presently unknown) such as housing density, environmental conditions, nutrition, and genetic lines could affect lateral skeletal integrity.
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Figure 2. Relationship between left and right apparent transverse humeral speed of sound (m/s) in postmortem specimens at 66 wk of age.
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Of particular interest is the robust relationship found between apparent humeral speed of sound (m/s) in live hens and subsequent postmortem evaluation in excised cleaned bones (Figure 3, R2 = 0.80, P < 0.0001), suggesting that tissue variable thickness of musculature and skin surrounding the humerus in live birds created little interference.
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Figure 3. Relationship between in vivo and ex vivo apparent transverse humeral speed of sound (m/s) at 66 wk of age.
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However, QUS still poses many limitations. First, it is not possible to standardize the depth of ultrasonic wave propagation along the humeral diaphysis, thus complicating the interpretation of derived data. There was very low correlation observed between QUS and humeral bone breaking force (R2 = 0.018, P > 0.05 Figure 4). Variable amounts of medullary bone may be present in the humerus and will be undetected by QUS, and this perhaps contributes to poor correlation with measurement of bone strength. According to Fleming et al. (1998), the medullary component of the humerus contributes to bone strength, resulting in increased resistance to fracture. If this is the case, then this may partially explain the poor relationship observed between the transverse speed of sound of primarily cortical bone with humeral breaking force, which takes into account the fracture resistance associated with the amount of cortical, cancellous, and medullary bone in the midpoint of a humerus. Hester et al. (2004) used dual energy x-ray absorptiometry to study changes in skeletal integrity in commercial laying hens and also suggested that any structural losses that occur with age in the tibia, and perhaps humerus, might be concealed by the continuous deposition of medullary bone. However, to substantiate this theory, it is important to be able to examine the region of interest in live birds with a 3-dimensional perspective and an enhanced resolution that is capable of separating different bone compartments to assess the degree of degeneration among different bone types (compact, cancellous, and medullary bone) over time. Fleming et al. (1998) reported that as hens age to 70 wk of age, the medullary bone volume increases at the same time that cancellous bone decreases in the proximal tarsometatarsus.
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Figure 4. Relationship between apparent transverse humeral speed of sound and humeral bone breaking force at 66 wk of age.
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Further investigations are needed to assess the value of QUS to indicate fracture risk. Siegel et al. (1958) performed observations on fractured rabbit tibias and found no relationship with QUS measurements. Even immediately after fracture, no effect on the ultrasound velocity was seen. Lowet and Van der Perre (1996) suggested that this effect might be caused by soft tissue swelling, affecting total propagation time, and by an inaccurate determination of the wave path length. There is also the need to account for any callus formation in response to fracture healing. Currently, it is not known if the calculation of ultrasound velocity will differ in a uniform callus vs. a geometrically irregular callus. For instance, Lowet and Van der Perre (1996) have recorded significant problems when comparing in vitro results from both of these types of calluses in human bones.
Results indicate the QUS is capable of detecting differences in humeral speed of sound (m/s) that are repeatable over time and correlate with classical in vivo measurement of bone density. The application of QUS to practical poultry production systems cannot be recommended until many key variables associated with its use can be more clearly defined. First, custom-designed probes are needed to accommodate the smaller bones found in chickens. This is important, because tissue thickness is a significant limitation in QUS. This situation limited the assessment of skeletal integrity of the humeri with this technique. Second, methodologies for testing ultrasonic wave path will have to be carried out in future studies to support and validate these concepts. The lack of knowledge of the wave path in the studies described in the current work somewhat confound interpretation of the results. It is also questionable whether clinical decisions can be made based on QUS alone. High-resolution, 3-dimensional assessments in live birds should be used to assess the effect of architectural integrity on apparent transverse speed of sound (m/s) of intact, healing, and freshly fractured bones. A suitable architectural appreciation in live birds will provide a visual diagnosis to evaluate the sensitivity of ultrasound for changes to the bone geometry, changes in bone material properties, or both.
Received for publication March 24, 2006.
Accepted for publication May 11, 2006.
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REFERENCES
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ABSTRACT
INTRODUCTION
