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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
* Department of Poultry Science, Texas A&M University, College Station 77843-2472; Department of Animal Sciences, University of Wisconsin-Madison 53706; and Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
4 Corresponding author: sricke{at}uark.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: skeletal integrity • alfalfa • bone mineral density • molting • bone strength
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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An induced molt using feed removal is a potential factor increasing structural bone loss in older laying hens. Structural bone loss in laying hens can increase fragility and susceptibility (Whitehead and Fleming, 2000). In the past, feed removal was the primary procedure used to induce a molt to stimulate a second egg-laying cycle in hens (Breeding et al., 1992; Bell, 2003; Park et al., 2004a,b). Typically, feed is completely removed for 10 to 14 d combined with a light reduction from 16 to 8 h (Brake, 1993). After a resting period of 0 to 21 d after feed removal, molted hens are fed regular layer rations to resume egg production. Several studies have indicated that induced molt using feed removal significantly decreased bone quality in laying hens (Garlich et al., 1984; Park et al., 2003; Kim et al., 2005, 2006; Mazzuco and Hester, 2005a,b). Garlich et al. (1984) reported that an induced molt using feed removal decreased femur weight and density in laying hens. Mazzuco and Hester (2005a,b) also reported that hens subjected to a fasting molt for 10 d exhibited a precipitous decrease in bone mineral density compared with nonmolted controls. Kim et al. (2006) indicated that induced molt using feed removal for 9 d significantly decreased bone dry weight, ash weight, breaking strength, mineral density, and mineral content compared with pretrial controls. After molted hens were fed maintenance diets or standard layer rations to stimulate egg production, bone qualities were slowly restored (Garlich et al., 1984; Mazzuco and Hester, 2005a). However, bone quality did not return to prefast levels, although bone quality increased when fasted hens were fed a standard layer ration (Newman and Leeson, 1999). It has been shown that a nonfasted molt using a wheat middling-based diet was less detrimental to bone mineralization than a fasted molt (Mazzuco and Hester, 2005b; Mazzuco et al., 2005). As a result of these studies and others, US egg producers who participate in the animal welfare-certified program of United Egg Producers (228 million layers) use only a non-feed withdrawal molting regime (United Egg Producers, 2006).
We developed alfalfa-based nonfasting molt diets that were comparable to postmolt performance of feed removal molting regimes (Donalson et al., 2005; Landers et al., 2005a,b) and in addition limited Salmonella Enteritidis colonization and support in vitro cecal microbial fermentation (Woodward et al., 2005; McReynolds et al., 2005, 2006; Dunkley et al., 2007). Alfalfa contains high protein with a good amino acid balance and is high in Ca with a slow rate of passage through the digestive tract (Sibbald, 1979; Sen et al., 1998; Garcia et al., 2000; Ponte et al., 2004). Recently, we reported that alfalfa-based molt diets may be beneficial in maintaining mechanical properties of bones during a 9-d molt period as compared with a fasted regime (Kim et al., 2007). In the present study, we evaluate the effects of alfalfa based nonfasting molt diets on skeletal integrity at 23 d postmolt.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Sixty White Leghorn hens, at 53 wk of age, were obtained from a nearby layer unit. The hens were placed in floor pens for 2 wk where they were taught to use nipple waterers, the type that would be available in the cages. At 55 wk of age, hens were placed in individual cages for 2 wk before initiation of treatments to be acclimatized to their new surroundings. They were given water and layer rations ad libitum, and production records were kept. One week before the initiation of the dietary treatments, the photo period in each of the rooms was changed from 16L:8D to 8L:16D. Hens were assigned randomly to 6 treatment groups: pretrial control (PC), full fed (FF), feed withdrawal (FW), 90% alfalfa meal and 10% layer ration (A90), 80% alfalfa meal and 20% layer ration (A80), and 70% alfalfa meal and 30% layer ration (A70). The negative control was the FW hens, which were not fed. The positive control consisted of the FF hens, which were given 100% layer ration. On d 0, ten hens were killed using CO2 inhalation for the PC group. Right femurs were collected for mechanical testing and histological analysis. After hens were subjected to different molting treatments for 9 d, they were fed a maintenance diet for 14 d. On d 23 postmolt, hens were killed using CO2 inhalation, and tibia, femur, and humerus bones were collected and evaluated using conventional bone assays, dual-energy x-ray absorptiometry (DEXA), mechanical testing, and histological analysis.
DEXA
To monitor bone integrity, frozen tibia and humerus with muscle and feathers intact were thawed and scanned using DEXA (Norland Medical System, Fort Atkinson, WI). Scanning began at the proximal end of each bone and took approximately 10 min for each bone. The orientation of the respective bone was the same for each scan. The bone mineral density and bone mineral content of the right tibia and humerus were determined from the DEXA scans (Schreiweis et al., 2003; Hester et al., 2004).
Conventional Bone Assays
Bone parameters were measured according to the methods described by Zhang and Coon (1997) and Park et al. (2003). After measuring bone mineral density and content using DEXA, bones were weighed in air and then reweighed while suspended in water at room temperature. Bone volumes were calculated assuming that the specific gravity of water is 1 g/cm3 at room temperature. For dry weight, the bones were initially dried at 100°C for 24 h and weighed. For ash weight, the dried bones were ashed at 600°C for 24 h, cooled in a desiccator, and weighed. Bone ash concentrations were calculated by dividing the ash weight of each bone by its volume (Zhang and Coon, 1997).
Bone Mechanical Properties
Bending Moments. To determine maximum bending moment, 3-point bending tests were conducted on the mid diaphysis of the right femurs using a 50-kg load cell with the Instron Machine Model 5566 (Engineering Corporation, Canton, MA). The support fulcra were adjusted (34 to 45 mm) depending on bone length. The yield load and ultimate load along with the deformation at each location were determined from the load deformation curve generated during the mechanical test. Loads were applied at 5 mm.
Staining Technique. Approximately 2 mm slices were removed at the bending point and stained using modifications of a differential stain for cortical and medullary bone in laying hens (Lynch and Maxwell, 1991). Before staining, the slices were defatted with a 3:1 ethanol:acetone solution for 60 min. Each defatted slice was subjected to 2 mL of the ethanol:acetone solution and 2 mL of water. After 10 min, 2 mL of additional ethanol:acetone solution was added and for every 10 min thereafter for a total of 60 min. After 60 min, bones were rinsed 3 times in distilled water. The slices were subsequently placed in acidified permanganate (equal parts of 0.3% aqueous potassium permanganate and 3% aqueous sulfuric acid) for 7 min and then rinsed 3 times with distilled water. They were placed in 5% oxalic acid for 7 min and then rinsed 3 times with distilled water. The slices were placed in 4.5% silver nitrate for 7 min and then in distilled water for 10 min. The slices were then again rinsed 3 times in distilled water followed by placement in ammonical silver for 7 min and rinsed in distilled water 3 times. The slices were then placed in a 10% formalin solution buffered in 0.85% NaCl for 7 min and then rinsed 3 times with distilled water. Finally, they were immersed in a 10% sodium thiosulfate solution for 10 min and rinsed in distilled water 3 times. After staining, slices were shielded from exposure to light and stored at – 20°C.
Embedding in Wax. The stained bone slices were thawed and embedded in orange wax. In a Precision Scientific Shaking Water Bath 50 (Precision Scientific, Chicago, IL), Distlefink Designs Candle Magic Wax Crystals (Craft House Corporations, Toledo, OH) were melted at 67°C in polystyrene culture test tubes. Bone slices were added to the melted wax, and any bubbles in the wax were removed by tapping the test tubes. After the wax had hardened, the test tubes were broken. The wax on one side of the slice was removed by running it across a piece of metal that was warmed to 58°C. Pictures of the waxed slices were taken with the Nikon Coolpix 500 digital camera (Nikon Inc., Melville, NY) through the lens of a dissection microscope.
Calculation of Bone Stress. Wax was deleted from the picture, and the pictures were edited using PhotoImpression from Nikon and Photoshop from Adobe (Adobe Systems Inc., San Jose, CA). The pictures were analyzed for moment of inertia as well as C value (the distance from the neutral axis to the extreme outer fiber) with and without medullary bone on 48 of the bones using the program Area Properties 3.6.1 (http://www.down-load.com/Area-Properties/3000-2260_4-10004867.html?cdlPid=8959582). From data collected from Instron and area properties, bone stress was calculated as follows:
where length = the length of the bone; C = the distance from the neutral axis to the extreme outer caliber; and moment of inertia is equal to the resistance of the bone to movement (Crenshaw et al., 1981).
Statistical Analysis
All data were subjected to a 1-way ANOVA as a completely randomized design using the GLM procedure of SAS (SAS Institute, 2001). Significant differences among means were determined by using Duncan’s multiple-range test at P 
0.05. Correlations of bone parameters were evaluated by Pearson correlation procedures (SAS Institute, 2001).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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. The FF hens exhibited significantly greater feed and Ca intakes compared with the A90, A80, and A70 hens (Table 1). Hens consuming the A80 or A70 molt diets had greater feed and Ca intakes than the hens consuming the A90 molt diet (P 0.05), whereas there were no significant differences in feed and Ca intakes between the A80 and A70 molt diet-fed hens. Feed and Ca intakes increased as the ratio of layer ration to alfalfa increased.
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). The FF hens produced eggs throughout the entire experiment period (data not presented). The FW, A90, A80, and A70 hens had significantly greater BW loss compared with the FF hens. The FW hens lost more BW than the A80 and A70 hens (P 0.05), but not the A90 hens. The percentage of BW loss exhibited a similar trend to the BW loss. The FF hens had significant lower percentage of BW than the FW, A90, A80, and A70 hens. The percentage of BW loss of the A70 hens was less than that of the FW hens (P 0.05; Table 1). These results are in agreement with Donalson et al. (2005). They indicated that as the ratio of layer ration to alfalfa increased, feed intake increased, and percentage of BW loss decreased during the 9-d molt induction period. The BW loss is likely a reflection of lowered feed intake. The lower feed intake associated with alfalfa could contribute to the low palatability of alfalfa by hens due to high levels of saponine (Matsushima, 1972). This component may suppress feed intake of molting hens, resulting in greater BW loss during a 9-d molt period in the present study.
Effects of dietary treatments on tibia and humerus parameters on d 23 postmolt using conventional bone assays are shown in Table 2. Tibia fresh weights of the FW and A90 hens were significantly lower than the PC hens. There were no significant differences in tibia volume among the treatments. The tibia dry weight of the FF hens was lower than the other treatments (P 0.05), whereas there were no significant differences among the PC, FW, A90, A80, and A70 hens. The FF, FW, A90, and A80, but not the A70, hens exhibited lower tibia ash weight compared with the PC hens. The tibia ash concentrations of the FF, A90, and A80 hens were lower than the PC hens (P 0.05), whereas there were no significant differences among the PC, FW, and A70 hens. For humerus parameters, the FF hens exhibited significantly lower humerus fresh weight, dry weight, and ash weight than the PC hens. The humerus fresh weights of the FW, A90, and A80 hens were lower than the PC hens (P 0.05). The A90, A80, and A70 hens exhibited significantly lower humerus dry weights compared with the PC hens. There were no significant differences in humerus ash weight among the PC, FW, A90, A80, and A70 hens. Overall, feed withdrawal and alfalfa molt diets decreased bone parameters measured by conventional methods during the 23-d postmolt. However, the hens fed the A70 alfalfa molt diet exhibited comparable bone parameters to PC hens. Because the hens fed the A70 diet had greater feed intake and Ca intake (Table 1), they appeared to maintain better bone quality compared with those fed an alfalfa molt diet (A90) or feed withdrawal hens.
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). These results also indicate that bone mineral content and density are decreased during a 23-d postmolt period regardless of molt dietary treatments.
Bone mineral density and content measured by DEXA are highly correlated with conventional bone assays (Table 3). Bone mineral density and content were positively correlated with fresh weight, ash weight, and ash concentration. Bone mineral density and content were highly correlated with each other (P < 0.0001). Tibia dry weight was positively correlated with ash weight and ash concentration (P < 0.0001). These results are in agreement with other studies (Zhang and Coon, 1997; Kim et al., 2005; Mazzuco and Hester, 2005a). Kim et al. (2005) reported that dry bone weight, ash weight, and ash concentration were highly correlated with each other. Mazzuco and Hester (2005a) indicated that a strong correlation existed among bone mineral content, bone mineral density, and bone ash weight responses.
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). Modulus of elasticity of the PC hens was greater than that of the FF and A90 hens. There were no significant differences in modulus of elasticity among the PC, FW, A80, and A70 hens. Moment of inertia of different bone compartments had no significant differences among dietary treatments. These results indicate that the FW, A80, and A70 hens have some beneficial effects on mechanical properties of bones during molt, whereas A90 and FF hens have poor mechanical properties of bones when compared with PC hens. Previously, we also observed that alfalfa molt diets exhibited retained mechanical properties of bones compared with the PC hens during a 9-d molt period (Kim et al., 2007). In the present study, the FF hens had poor bone parameters compared with the PC hens or the other dietary treatments. Because the FF hens had the same decreased light schedule as the other molting hens, they had much less time to access feed than the PC hens, but they continued to lay eggs throughout the molt periods, resulting in more bone resorption for the eggshell formation and decreased bone mechanical properties.
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) and cortical bone (Figure 2) increases (P < 0.02), whereas no significant relationship was detected between moment of inertia of medullary bone and bending moment (Figure 3). These results indicate that cortical bone affects mechanical strength of bone, but medullary bone does not. Although there are conflicting conclusions on medullary bone and bone strength by different studies, our results are in agreement with other studies (Knott et al., 1995; Knott and Bailey, 1999), which indicate that medullary bone does not contribute to mechanical strength of bone.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Department of Poultry Science, University of Georgia, Tifton, GA 31793-0478.
3 Current address: Center for Food Safety-IFSE and Food Science Department, University of Arkansas, Fayetteville, AR 72704-5690.
Received for publication January 18, 2008. Accepted for publication June 13, 2008.
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REFERENCES |
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