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METABOLISM AND NUTRITION |
* Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada, T6G 2P5; and School of Animal Studies, University of Queensland, Gatton, Australia, 4343
1 Corresponding author: doug.korver{at}ualberta.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: broiler chicken • indicator amino acid oxidation • bicarbonate retention factor • quantitative computed tomography • bone density
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Tabiri et al. (2002a) reported the bicarbonate retention of broiler breeder roosters between 24 and 36 wk of age. However, studies have shown that factors such as age and metabolic state have an effect on bicarbonate retention (Armon et al., 1990; Barstow et al., 1990). Therefore, the bicarbonate retention factor (BRF) reported by Tabiri et al. (2002a) may not be applicable to growing broiler chickens. Additionally, broiler chickens at different stages of development are likely to have different BRF due to changing metabolic rates (Barstow et al., 1990), which may be associated with changes in BW gain, bone growth, and muscle deposition.
Clugston and Garlick (1983), Tomera et al. (1983), and Hoerr et al. (1989) suggested that the most accurate method to account for CO2 label retention is to perform a retention study either concurrently (using 13C-labeled CO2 and 14C-labeled substrate or vice versa) with the oxidation study or to perform a retention study the previous day in the same individual under the same metabolic conditions. However, the generally accepted approach is to determine average retention values for similar subjects under the same metabolic conditions and apply those values to oxidation studies (Irving et al., 1983; Van Aerde et al., 1985; Benevenga et al., 1992; Tabiri et al., 2002a,b). Although the former approach is considered more accurate, it involves several drawbacks for studies on growing animals. These include the time and cost involved, as well as the stress the animal is subject to during the extra experimental days.
One of the main concerns of critics is that the bicarbonate infused into the bloodstream does not have the same starting point as bicarbonate that arises from cellular oxidation (Hamel et al., 1993). That is, infused bicarbonate enters the plasma pool, whereas bicarbonate from oxidation first enters the intracellular pool. The distribution of infused NaH14CO3 throughout body tissues has been investigated in several studies (Shipley et al., 1959; Poyart et al., 1975a; Tomera et al., 1983). Tomera et al. (1983) used perfused rat livers to show that bicarbonate retention represents the reincorporation of CO2 from substrate oxidation by the liver, indicating that bicarbonate retention studies do in fact accurately estimate the reincorporation of metabolically-produced CO2 into other metabolic products. This information, combined with the reduced number of experimental days, makes the determination of a BRF the more desirable approach.
Poyart et al. (1975a) found that 30% of the bicarbonate in the blood that is supplied to the bone is transferred via a labile bicarbonate pool. This pool is thought to be associated with the bone water rather than the structural bicarbonate, which has a much slower turnover rate. The amount of bicarbonate entering the labile bone water pool is believed to be limited by the blood flow to the bone rather than the capacity of the bone to sequester bicarbonate (Poyart et al., 1975b). Rapidly growing chickens have a high rate of bone formation (Applegate and Lilburn, 2002), which could result in a substantial amount of bicarbonate fixed through hydroxyapatite deposition (Whittow, 2000). Therefore, the effect of this potential bicarbonate pool should be studied before IAAO experiments in rapidly growing broiler chickens.
The present research was performed to develop a suitable BRF for correction of steady-state 14C-labeled substrate oxidation experiments on broiler chickens at weekly intervals from 7 to 42 d of age. To identify any potential effects of the stage of bone development on bicarbonate retention, bone traits were determined for birds across the same age range. The total trabecular and cortical bone mineral densities, as well as cross-sectional areas, were determined by quantitative computed tomography (QCT) at the midpoint of the femur and were used as an indicator of bone size and mineralization (Korver et al., 2004).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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All procedures were reviewed and approved by the University of Alberta Faculty Animal Policy and Welfare Committee and were in accordance with Canadian Council on Animal Care (1993) guidelines. Two hundred male Ross 308 chicks were obtained from a commercial hatchery on the day of hatch. The chicks were transported to the Metabolic Research Unit at the University of Alberta Edmonton Research Station and housed in a single floor pen with an initial stocking density of 11.4 birds/m2. Temperature was maintained according to an industry standard temperature schedule and adjusted according to bird behavior and age. Birds were provided feed and water ad libitum for the duration of the experiment. A commercial-type starter diet was fed from 0 to 21 d (21.4% CP, 3,000 kcal/kg of ME, 1.02% Lys), and a grower diet was fed from 22 to 42 d (19.6% CP, 3,096 kcal/kg of ME, 0.968% Lys). A lighting schedule of 23L:1D per day was used for the duration of the experiment.
Experimental Design
Birds were individually weighed weekly to determine the mean population weight. Beginning on d 0 and at weekly intervals to 42 d of age, 16 birds within 5% of the mean population weight were selected. Four of the selected birds at each age were randomly designated for use in the bicarbonate retention experiment, whereas the remaining 12 were killed via cervical dislocation for subsequent bone analysis by QCT. At the end of the bicarbonate retention portion of the study each week, the 4 birds used for IAAO were also euthanized and included in the bone analysis. As the QCT was done on bone samples excised from the euthanized birds, different birds from the same population were used for each age.
The 4 birds selected for the bicarbonate retention trial were placed in polycarbonate oxidation chambers and allowed free access to feed and water at all times. On d 0, 7, and 14, the chambers used were 30 cm x 30 cm x 25 cm; the flow rate of air through the chambers was 10 L/min. From 21 to 42 d of age, larger chambers, as described by Tabiri et al. (2002a), were used. Those chambers measured 60 cm x 40 cm x 50 cm; a flow rate of 20 L/min was used.
Before each oxidation study, each bird was given an accurately weighed priming dose (approximately 1.2 µCi of NaH14CO3; ARC 138C, American Radiolabeled Chemicals Inc., St Louis, MO) by oral gavage. The use of a priming dose allowed the rate of appearance of 14CO2 in the breath to reach equilibrium more rapidly (Allsop et al., 1978). After 15 min, the birds were given the first of 8 half-hourly doses of 0.5 µCi of NaH14CO3 in 0.25 mL of saline by oral gavage. The isotopically-labeled bicarbonate was mixed into a saline solution and stored at 4°C in a sealed glass bottle for the duration of the experiment to minimize liberation of the label as 14CO2 during the study (Wolfe, 1992). Radioactivity of the stock solution was determined on each experimental day before initiation of dosing. Syringes were filled with the appropriate amount of isotope solution; syringe weights were recorded immediately before administration of the dose. Because the labeled bicarbonate was applied in trace amounts, and therefore was assumed to have no significant effect on body-pool bicarbonate concentrations, the dosage applied was the same for all birds, regardless of BW. Oral doses were applied by attaching a piece of surgical Tygon tubing (Saint-Gobain Performance Plastics Corp., Courbevoie, France) to the syringe, inserting the tubing into the crop, and gavaging the isotope solution. The syringe was weighed again after the oral dose was administered to determine the precise amount of isotope that was given. To administer the oral dose, each bird was removed from the chamber. At the same time, the feeder was removed and weighed to determine the half-hourly feed consumption. This procedure took approximately 20 s and likely did not significantly affect the collection of 14CO2. Collection of expired CO2, determination of 14C in breath, and calculation of bicarbonate retention was determined as described by Tabiri et al. (2002b).
Each of the 16 birds sampled on each experimental day was killed by cervical dislocation, and the right femur was removed. The femur from each bird was immediately analyzed by QCT using a Norland Stratec XCT (XCT Research SA, Norland Corp., Fort Atkinson, WI; Korver et al., 2004). Briefly, an initial longitudinal scan was performed on each femur. From this image, the midpoint of the bone was determined, and a cross-sectional scan was performed at that point. The cross-sectional scan quantified the femur bone mineral density and cross-sectional area of cortical, trabecular, and total bone of the 1-mm slice. Calcium content of the femur increases proportionately to total skeletal Ca content (Itoh and Hatano, 1964), thus the QCT measurement gives an indication of changes in overall bone mineralization
Statistical Analysis
Plateaus in percentage of dose retained were determined using linear regression (SAS Institute, 1999), with a line having a nonsignificant slope (P > 0.10) and a CV<15% considered a plateau. Differences in mean 14C retention, bone mineral densities, and areas at different ages were analyzed using the pdiff option of the PROC GLM function of SAS (SAS Institute, 1999). Correlations and regressions were performed using the PROC CORR and PROC REG functions of SAS software, respectively. Differences betwen means were considered significant at the P < 0.05 level. The effect of age on BRF, using BW as a covariate, was determined using the PROC GLM function of SAS (SAS Institute, 1999).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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) fell within the range described in the literature for broiler breeders (14%; Tabiri et al., 2002a), adult pigs (19.0 to 21.2%; Moehn et al., 2004), and other species (El Khoury et al., 1994); however, the retention factors for d 0, 14, 21, and 28 (BRF of 47.2 ± 2.04, 56.0 ± 5.38, and 62.9 ± 2.63%, respectively) were higher than previously reported for other species. Experiments in other animals have resulted in a wide range of retention values, ranging from 21% in dogs (Downey et al., 1986), 22 to 26% in neonatal pigs (Benevenga et al., 1992), and 6 to 48% in humans (Hoerr et al., 1989; El Khoury et al., 1994).
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. Body weight group clusters on Figure 1 represent the age groups from 7 to 42 d. Within the BW range of 100 to 1,900 g, the equation Y = 6.95 x 10–2X –3.51 x 10–5X2 + 27.58, where X = BW and Y = bicarbonate retention, describes the relationship between X and Y (P < 0.0001, R2 = 0.79). Birds larger than 1,900 g showed a constant BRF of 35.15 ± 1.095% (mean ± SEM). The BRF data for d 0 were excluded from the regression because of a large degree of variability in BRF among the birds. The variability was likely due to differences in water consumption of the newly-arrived chicks and, therefore, in hydration and pool size among the chicks. In addition, the relationship between bicarbonate retention and age was tested (P < 0.01, r2 = 0.80) but did not approach the accuracy of the prediction equation based on BW. The equation shown on Figure 1 can be used to accurately predict BRF of broilers using the BW of the bird. The BRF reached an apparent plateau at 1,900 g of BW (35 d of age); therefore, the average value of 35.15% bicarbonate retention is recommended for birds from 1,900 to 2,700 g. The use of this equation in future IAAO studies assumes that the experimental diet will not significantly alter the physiological state of the bird and that the dietary treatment will not affect the metabolic processes that result in the fixation or retention of bicarbonate.
Cortical, trabecular, and total femur cross-sectional area increased with age to 42 d of age, with cortical bone area showing the largest proportional increase during this time (Table 1). Both cortical and total bone cross-sectional areas nearly doubled each of the first 4 wk. After 28 d, weekly proportional bone growth (cross-sectional area) was somewhat reduced, only increasing by a factor of about 1.5. Similarly, cortical cross-sectional area of the tibia of broilers increased most rapidly from 1 to 28 d of age, after which the rate of increase slowed (Leterrier et al., 1998). Williams et al. (2000) reported that broiler tibiotarsus cortical bone width increased rapidly to 18 d of age, at which point it did not increase further. The growth in cross-sectional area was roughly proportional to the increase in BW of the birds from d 7 to 42. Applegate and Lilburn (2002) reported that BW differences accounted for more than 98% of the variability in femur and tibia bone weight.
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The continued increase in area and decrease in total and trabecular density suggests that the rate of bone deposition as a proportion of BW decreases after 28 d, which may partially explain the reduction in BRF. Similarly, bone density of the tibia of broiler chickens growing rapidly or slowly reached a peak at 12 d of age and remained constant to 42 d of age; defatted, hydrated tibia weight increased to 26 d of age, after which further increases were minimal (Leterrier et al., 1998).
The femur was chosen because the mineralization of this bone is more representative of skeletal mineralization than any other bone. Itoh and Hatano (1964) reported that changes in overall skeletal mineralization of White Leghorn chicks from 0 to 3 wk of age was most accurately reflected in femur mineral content. The pattern of bone growth as determined by QCT analysis of a femur cross-section is supported by other research in broiler chicks, in which the rate of tibia (Bond et al., 1991; Skinner and Waldroup, 1995; Rath et al., 2000), tibiotarsus (Williams et al., 2000), and femur (Bruno et al., 2000; Applegate and Lilburn, 2002) growth decreased after 18 to 28 d of age in broilers.
Studies by Poyart et al. (1975a, b) in rats showed that bone acts as a bicarbonate pool. These authors showed that 30 to 50% of the bone CO2 stores are dissolved in water associated with the bone tissue and can be exchanged with blood CO2. The remaining 50 to 70% of bone CO2 is associated with the crystalline structure of the bone and will exchange with the aqueous portion at a very low rate. They also showed that the water associated with the bone declines with age (Poyart et al., 1975a,b). The decreasing rate of skeletal growth and mineralization past 21 d of age in broiler chickens (Bond et al., 1991; Skinner and Waldroup, 1995; Bruno et al., 2000; Williams et al., 2000; Applegate and Lilburn, 2002), as well as reductions in bone moisture content (Yalcin et al., 2001), could explain the rapid decrease in bicarbonate retention. Because the largest loss of bicarbonate associated with the bone is due to exchange with this water, a decrease in the water associated with the bone would result in a decrease in the proportion of bicarbonate that is sequestered.
Poyart et al. (1975b) found that, in rats, 30% of the CO2 in the blood that was partitioned to bone tissue was retained in the bone. The amount of bicarbonate sequestered in the bone was limited by blood flow to the tissue rather than the exchange rate. Because the fractional blood flow that reaches the skeleton in chickens is not known, the potential contribution of this slowly exchanging pool cannot currently be accurately estimated. As bone cross-sectional area increased more rapidly than BW from 14 to 28 d of age (Table 1), it would be expected that a higher proportion of the bicarbonate pool would be sequestered during this period than during periods of relatively slower bone development due to an increase in the volume of water associated with bone tissue. Whereas Poyart et al. (1975a) described a reduction in water associated with the bone of rats as a percentage of the total bone weight of about 1.7% in 12- vs. 10-wk-old rats, the increase in relative bone weight associated with the changes in BW and bone cross-sectional area seen here would result in an increase in total bone water.
The relatively high BRF found at 14, 21, and 28 d may be explained by the pattern of bone development of the chick described earlier. As broilers grow very rapidly from 0 to 42 d, the incorporation of labeled bicarbonate into bone pools may contribute significantly to the relatively low recovery of labeled, infused bicarbonate. Assuming the femur can be taken as representative of total body bone growth (Applegate and Lilburn, 2002), this high growth rate of bone could partially account for the high BRF found in 14- to 28-d-old birds. The correlation (r = 0.50, P = 0.007) between bicarbonate retention and total bone density shows that 25% of the variability in bicarbonate retention was associated with changing bone density with age (Table 2). This helps to explain the relatively high values for retention found in young, rapidly growing birds. The total bone density for the broilers was highest at 21 and 28 d. The measurements of bone area and density were made at the midpoint of the femur, with the assumption that this measurement is proportional to the development of the rest of the skeleton. Applegate and Lilburn (2002) have shown that the femur is more representative to total bone development than the tibia. Their study also showed that femur length reached a plateau at 35 d of age, and epiphyseal and diaphyseal ash content reached a plateau at 21 d. This indicates that bone growth at the growth plates begins to slow from 3 to 5 wk of age, which agrees closely with the time when bicarbonate retention decreased in the present study. The probability that bone growth affects bicarbonate retention should be considered in any labeled C research using rapidly growing young animals.
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, with an R2 value of 0.79, is sufficiently accurate for birds having BW from 100 to 1,900 g. The total bone density, as determined by QCT, was shown to account for 25% of the variation in bicarbonate retention (r2 = 0.25, Table 2). Studies involving different types of chickens, such as laying hens, broiler breeder pullets, or broilers having BW outside the described range, would require additional study to determine the appropriate BRF. The results of this experiment require the assumption that changes in future experimental diets do not significantly alter the fixation of bicarbonate or the bird’s ability to sequester bicarbonate in body pools. Finally, these data have application to research in other animals, specifically, that to obtain correct BRF, rapid bone growth should be considered a significant source of bicarbonate retention.
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ACKNOWLEDGMENTS |
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Received for publication February 15, 2006. Accepted for publication June 24, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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