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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
* Faculty of Engineering, Department of Biological and Agricultural Engineering, Department of Veterinary Pathology, and Department of Avian Medicine, University of Georgia, Athens 30602
1 Corresponding author: tfoutz{at}engr.uga.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: biomechanics • mobility • tendon • lameness
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Weeks et al. (2000) indicated that the increased growth rate of meat-type chickens has changed the behavior of birds. For example, broilers now spend 76 to 86% of their time sitting. In mammalian studies, physical inactivity or immobilization decreases collagen remodeling and adaptation (Kjaer, 2004) and adversely affects the biomechanical properties of tendon (Almeida-Silveira et al., 2000; Palmes et al., 2002). Studies involving chickens have found similar results when activity is related to altered proteoglycans synthsis in the gastrocnemius tendon (Yoon et al., 2004) and has an effect on the overall biomechanical performance of the musculoskeletal system of the leg (Griffin et al., 1998).
The purpose of this study was to investigate the relationship between broiler activity and tendon structural integrity. The working hypothesis was that increased mobilization of broiler chicken leads to increased strength and improved biomechanical performance of the gastrocemius tendon.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Commercial, female Arbor Ross broiler chickens were used as test subjects. This particular strain was chosen due to the large physical size of the birds, and gender selection was based upon evidence that female behavior to the mobility treatment (treadmill pacing) used in this study was more consistent than male behavior (Griffin et al., 1998; Yalcin et al., 1998). The test population was acquired at 1 d of age and was raised in environmental chambers located at the Driftmier Engineering Center at the University of Georgia. Samples were taken from the test population beginning at 3 wk of age and ending at 6 wk of age. This permitted investigation of the development of the skeletal system through the normal life span of a commercially produced food animal. The housing and mobility procedures reported herein were approved by the University of Georgia Institutional Animal Care and Use Committee (A970154).
Husbandry
One-day-old chicks were placed in the environmental chambers that were held at 29°C (±1°C) and low humidity. During the first week, brooders were used to provide supplemental heat. The flooring consisted of 76 mm of wood chips covering a concrete pad. For the first 2 wk, all birds were fed a broiler starter diet, and then all birds were switched to a broiler grower diet until the study was terminated. In addition, all subjects were given unrestricted access to autoclaved, medicated feed: 12.5 g/ ton of bacitracin and 90 g/ton of monensin (Foutz et al., 1997). Studies have shown that supplementing chicken feed with monensin (150 g/ton) has no effect on leg integrity (Rath et al., 1998). This is significant, because in vitro cell studies have utilized monensin, a Ca ionophore, as a causative agent to prohibit procollagen secretion (Ledger et al., 1980; Satoh et al., 1996; Li et al., 1997). Monensin did cause an increase in soluble collagen fractions in avian tendon, but additional factors, such as heat, were determined to be necessary to affect a significant change in tendon physiology (Rath et al., 1998).
Viral Screening
Weekly blood samples were drawn from each flock and submitted for ReoELISA at The University of Georgia Poultry Diagnostic Research Center. If, at any testing, birds tested positive for general reovirus infection, the entire flock was terminated, the environmental chambers were disinfected, and a new flock was started. Only samples from reovirus-negative flocks were analyzed for this study.
Experimental Groups
The treadmill treatment shown by Yoon et al. (2004) to alter proteoglycans synthesis in the gastrocnemius tendon of chickens was used in this study. Beginning at 1 d of age, all birds were placed in the treadmill for acclimation. This acclimation was deemed necessary based on a review of previously published studies (Brackenbury et al., 1990; Bain, 1998). At 7 d of age, all birds were reintroduced to the treadmill by pacing at 0.22 m/s for 5 min/d. This was done to make all the birds comfortable with the treadmill pacing. Over the course of the next 2 wk (from age 1 to 3 wk), the speed of pacing during the 5-min acclimation period was gradually increased from 0.22 to 0.45 m/s. Birds that were determined to be unwilling to walk on the treadmill were removed from the population.
Treatments.
After the acclimation period, the birds were divided into 2 groups, control and treadmill-pacing (Table 1
). Control birds were selected from a population of birds willing to walk on the treadmill as described above. At the start of the treatment, 3 wk of age, these control birds were no longer exposed to the treadmill.
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The birds were paced at approximately 0.45 m/s, which corresponds to 50% of the speed at which the birds will attempt to fly (Biewener et al., 1986). During the initial treadmill-pacing session, the performance of each bird was monitored to confirm that 0.45 m/s was the proper speed for that particular bird. If during the treadmill-pacing routine, a bird refused to walk or was behaving in a manner that could cause harm to the bird, it was removed from the treadmill for that particular session. The total time that each bird actually walked on the treadmill was recorded daily and used to quantify the treadmill performance of each bird. Treadmill performance was calculated by dividing the number of total minutes that the bird actually walked by the number of treadmill minutes that should have been walked.
Tissue Collection
Birds were removed at weekly intervals (3, 4, 5, and 6 wk of age) from each experimental group. Over the course of the study, 5 birds/treatment per weekly interval were sampled, and the experiment was repeated 3 times. In other words, tissue was collected from 15 birds/treatment per weekly interval. The birds were removed using a random selection routine.
Birds were killed with an overdose of CO2. One leg was used for biomechanical testing. The gastrocnemius muscle-tendon-bone (GMTB) was dissected and wrapped in towels soaked in isotonic avian saline, placed in a freezer bag, and then stored at –70°C until quasistatic failure testing.
Bird Growth
Normal growth was monitored by measuring bird body mass, shank lengths, and shank widths every 7 d, beginning at 1 d of age. For the first 3 sampling sessions (1 d, 1 wk, and 2 wk of age), an average body mass was determined for the entire flock, because wing-banding was not possible. Body mass collected at subsequent time points (3, 4, 5, and 6 wk) was recorded for individual birds based on wing band identification. Shank length was defined as the distance from the footpad to the tibiotarsal joint. Shank width was defined as the largest diameter of shank at a distance equal to 50% of the length of the shank.
Gastrocnemius Tendon Midregion Cross-Sectional Area
Before the quasistatic tensile testing, the GMTB complex was removed from –70°C storage, thawed at room temperature, and placed in a physiologic saline bath (0.85% saline, 38.9°C). Using digital calipers (MaxCal, Fowler Co. Inc., Newton, MA), the gage length (mm) of the gastrocnemius tendon was recorded, and the midpoint was marked. Overall gage length was recorded for each tendon by measuring from the bony insertion to the top of the bifurcation. Based on the gage length measurement, the middle third of the tendon was determined and used as the tendon control area for quasistatic testing. Tendon width and thickness were recorded at the proximal, midpoint, and distal points of this control area (Figure 1). Using these width and thickness measurements, the traverse midregion cross-sectional areas were calculated at 3 distinct points that were marked as P, M, and D.
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Biomechanical evaluation of tendons provided an indication of the strength changes that result from structural alterations. Smith et al. (1996) has shown that quasistatic testing of previously frozen tendons did not significantly alter ultimate tensile strength, failure strain, and toughness; however, stiffness values were affected by prior freezing. The GMTB was loaded in the Instron 4201 Material Tester (Instron Corp., Norwood, MA) by clamping the gastrocnemius muscle and the metatarsus bone, which left the tendon free of the clamping mechanism (Mohammed et al., 1995). Basically, the tester deformed the tendon, and an electronic load cell recorded resistance of the tendon to that deformation. The tendon control area was defined as the area of interest used to determine the biomechanical properties of the tissue (Figure 1
).
When clamping soft tissue to the tester, damage will typically occur and thus change the biomechanical behavior of the tissue. Matthews et al. (1996) found that cryoclamps can be used to overcome these effects. In the study herein, customized cryoclamps were used (Figure 2). Tendon sample was dissected from the leg such that the tendon remained attached to the gastrocnemius muscle and the metatarsus bone. The gastrocnemius muscle tendon interface up to the bifurcation was placed in 1 cryoclamp, and the tendon-bone insertion up to the tibiatarsus joint was placed in the other cryoclamp. The design of the cryoclamps allowed the tendon control area of interest to be kept at a temperature between 24 and 30°C (Figure 3). By keeping the tissue area near the clamps close to freezing and the tendon control area warm, tendon deformation and failure due to loading was within the control area.
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A phenomenological model (Henry et al., 2000) was applied to the loading curve of each tendon, and model parameters were used to calculate maximum relaxation load, maximum relaxation stress, maximum relaxation deformation, and maximum relaxation strain. The biomechanical parameters of toughness, tangent modulus, and secant modulus (Beer et al., 2002) were also calculated. Two forms of toughness, structural toughness and material toughness, were determined. Because this study was dealing with soft tissue that produces a nonlinear loading curve, the tangent modulus was based on the slope of the linear portion of the loading curve, and the secant modulus was based on the slope of line connecting the origin of the loading curve to a point on the curve that represents the maximum load applied (Figure 4).
The biomechanical parameter maximal tensile strength was measured as the maximum stress withstood by the tendon, where this stress is maximum load divided by the original cross-sectional area of the tendon at the mid-region cross-sectional area.
Morphometric Measurements
Tendon Notch.
During the quasistatic tensile testing, a trend in tendon geometry was noted. Birds in the treatment group exhibited increased tissue width, "notches," at the level of the tibiotarsal joint and well within the region of interest (Figure 1). Using the video dimension analyzer, the location and width of the notch was measured for each tendon tested.
Statistical Analysis
Effect of treatment on the collected data was analyzed using the GLM procedure (SAS Institute, 1990). A split-plot design using treatment and repetitions as plots was utilized. The treatment was the treadmill walking. Three repetitions were done. Five birds per repetition were sampled from the control group and treatment group when the birds were 3, 4, 5, and 6 wk of age. Significance of effect was accepted at P 
0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The treadmill treatment had a significant effect on body mass (Table 2) after 2 wk of the treatment. A significant effect of treatment (P 0.05) was found at 6 wk of age for measurements of shank length and at 5 wk of age for measurements of shank width. No other effects due to treadmill treatment were found for maximum relaxation, relaxation tensile load, or other relaxation structural and material properties (Table 3). The treadmill pacing treatment had no effect on structural toughness or on the material toughness parameters (Table 4). Treatment did not have an effect on the structural or material tangent modulus of the gastrocnemius tendon (Table 5), although the moduli of the control tendons were consistently higher than the structural and material moduli calculated for the treadmill-paced tendons.
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) or on the tensile deformation at failure. Gastrocnemius tendon midpoint thickness and width was used to normalize tensile load at failure. By normalizing tensile load at failure to these geometric measurements, it is possible to reduce the effect of variance in specimen size. Normalizing maximum tensile load by the control area midpoint thickness and by the control area midpoint width did not reveal a significant effect of treatment. The tensile stress at failure and tensile strain at failure (Table 6) also did not indicate a significant effect of treatment.
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When the various components of the response curve were broken down, the treadmill-paced group exhibited a lengthening of the toe region of the biomechanical loading response curve. For the treadmill-paced birds, the toe region was expanded as well as the failure region. This provides evidence that the pacing exposure could have increased tendon compliance of the treadmill treatment group. Current literature reports that tendon strength lies in large-diameter fibrils and crosslinking (Matthew and Moore, 1991; Bailey et al., 1998; Benjamin and Ralphs, 1998). However, if the application of exercise increases compliance without losing strength, as seen in this study, it is possible that the trained tendon can accept greater physiological loading without greatly increasing the stress in the tissue. A decrease in stress would reduce the chance of microdamage, which could eventually weaken the tissue and provide opportunity for failure.
The relaxation region of the loading curve represents the uncrimping of fibers and the elastic response of the tissue, an extension of this zone may indicate that the treadmill treatment increases the compliance of the tendon compared with control birds. Increased compliance would allow the tissue to accept more deformation and diffuse loading; however, it does not necessarily confer additional strength characteristics to the tendon. Therefore, a treadmill-paced tendon could exhibit more elastic behavior and fail at the same load as its control counterpart, as evidenced by the nearly identical values of relaxation deformation. The explanation for increased compliance could lie in a reorganization of the matrix. If the treadmill pacing treatment caused a diminishment of the reducible crosslinks in the fibril hierarchy, a more compliant tendon would result at the cost of structural integrity. From the material properties standpoint, neither the stress nor strain experienced by the sample tendons in the control or treadmill-paced treatment showed any appreciable differences with the treatment. Whether this is due to a normally dynamic growth spurt of the birds in the age range investigated in this study or is an indication that the intensity of the pacing was not sufficient to bring about a change remains to be determined.
In this study, no age-related effects on the structural or material modulus of the juvenile avian gastrocnemius tendon could be appreciated in the control or treadmill-paced groups. Structurally, the modulus of the control and treatment tendons was similar; however, the material tangent modulus reveals a consistent trend of decreased stiffness with the treadmill pacing treatment. This result would agree with the findings of relaxation and failure deformation in that a more compliant tendon would not exhibit a higher degree of stiffness in comparison. This finding also agrees with biomechanical tests of adult rat-tail tendon from paced rats (Viidik et al., 1996); however, in a similar test of Achilles tendon from treadmill-exercised rats, no significant changes in stiffness were found (Nielsen et al., 1998; Skalicky and Viidik, 2000). Buchanan and Marsh (2001) showed that 8 wk of treadmill pacing increased the stiffness of the Achilles tendon of the guinea fowl. The incongruity between this particular investigation and the results of Buchanan and Marsh (2001) could lie in the duration of the exercise exposure, level of intensity of the exercise, or in a combination of the 2 factors. Microdamage occurring as a result of high intensity exercise would translate to adhesions in the healing tendon, which would increase the stiffness. Because the differences were noted in the material tangent modulus, it seems appropriate that the primary focus for investigation of this response would be aimed at the matrix constituents and possible changes in matrix organizations (i.e., crosslinking of collagen fibers).
During loading curve data collection with the VDA, researchers noticed that treatment tendons appeared to have an increased amount of tendon tissue or notch (Figure 1). Statistical analysis indicated no correlation between the notch size and treatment; however, approximately 33% of the treatment tendons had the notch as compared with <10% of the control tendons. Furthermore, of the notched tendons, the treadmill-paced group had consistently more tissue in the notched region.
Although not statistically significant in this study, the presence of a notch of tendon tissue at the level of the tibiotarsal joint in one-third of the gastrocnemius tendons of the paced group may provide an illustration of how the tissue responds to the loading condition. Further investigation is warranted to determine the orientation of collagen fibrils within this area and to determine how the loading is transmitted through this region. In addition, Yoon et al. (2004) showed quantitative changes in proteoglycans in juvenile gastrocnemius tendons from treadmill-paced chickens: an increase in hyaluronic acid and decorin levels and a decrease in aggrecan and keratan sulfate with exercise. Another interesting note was that all of the notches found on the tendons occurred just after the bifurcation. Whether this is significant or indicative of a modeling response to applied load remains to be determined. Few morphometric changes were noted in the gastrocnemius tendons of the treadmill-paced birds as compared with control birds. The most notable was the presence of a tissue notch on 33% of the tendons in the treadmill-paced group. In all instances, the notch was located within the midregion of the tendon at the level of the hock joint.
Received for publication August 4, 2006. Accepted for publication September 9, 2006.
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