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Using corn starch as basal diet to determine the true metabolizable energy of protein feedstuffs in Chinese Yellow chickens
Research Notes Using corn starch as basal diet to determine the true metabolizable energy of protein feedstuffs in Chinese Yellow chickens L. Q. Ren,* H. Z. Tan,† F. Zhao,* J. T. Zhao,† J. Z. Zhang,* and H. F. Zhang*1 *The State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China; and †Wen’s Foodstuffs Group Corporation Ltd., Guangzhou 527439, China ABSTRACT Two experiments were conducted to investigate the feasibility of using corn starch as the basal diet to determine the ME of protein feedstuffs using the TME assay in Chinese Yellow chickens. In the first experiment, the TME of corn starch were determined by force-feeding 25 or 40 g of feed. To test the repeatability of the bioassay, the same experiment was repeated 4 times. In the second experiment, the TME of soybean meal and cottonseed meal was determined by considering corn starch as the basal diet, while corn was fed alone to the chickens. To test the accuracy of the TME assay for individual ingredients, the additivity was evaluated by determining the TME of 3 mixed diets: corn-soybean meal diet, corn-cottonseed meal diet, and corn-soybean meal-cottonseed meal diet. In experiment 1, the value of endogenous energy loss was 16.76 to 18.46 kcal/48 h, and no significant differences
between the 4 assays were noted. The TME and energy metabolizability of the 25-g corn starch treatment (4.06 kcal/g and 98.06%) were higher than those of the 40-g treatment (3.79 kcal/g and 91.45%; P < 0.01); whereas the CV were less than that of the 40-g treatment, indicating that it is reasonable to use the TME value of the 25-g treatment in feed formulation. In experiment 2, the TME values for corn, soybean meal, and cottonseed meal were 4.02, 3.39, and 2.92 kcal/g, respectively. The observed and predicted TME values of the corn-soybean meal, corn-cottonseed meal, and corn-soybean meal-cottonseed meal diets were in high agreement with differences ranging from −0.02 to 0.01 kcal/g. None of the differences was significant, indicating an accurate measure of the TME of the individual ingredients. Thus, using corn starch as the basal diet to determine the TME of protein feedstuffs was validated.
Key words: basal diet, Chinese Yellow chicken, corn starch, protein feedstuff, true metabolizable energy 2012 Poultry Science 91:1394–1399 http://dx.doi.org/10.3382/ps.2011-01838
INTRODUCTION The Chinese Yellow chicken is a crossbreed of the recessive white hen and the Chinese-native Yellow rooster, and it is characterized by slow growth, light market weight, and good taste and meat quality. In the recent 5 yr, the annual total Chinese Yellow chickens were about 3 billion, which accounted for one-half of China’s total production of broilers (NPIA-CAAA, 2008). However, data are still scarce on the utilization of nutrients in feedstuffs for the Chinese Yellow chickens. Metabolizable energy is an important dietary energy utilization response criterion in feed formulation and represents a large portion of the total cost of the broiler industry. So, it is especially critical to select a precise and accurate method to determine the ME of feedstuffs in Chinese Yellow chickens. ©2012 Poultry Science Association Inc. Received September 1, 2011. Accepted November 19, 2011. 1 Corresponding author: [email protected]
Developed by Sirbald (1976), the accepted procedure for measuring the TME of feedstuffs has been widely used in more than 30 countries (Dudley-Cash, 2009). This method enables the single feedstuff to be fed in a precisely measured quantity, which is carried out rapidly and inexpensively. However, it was found that when protein feedstuff was tube-fed alone, the protein content was much higher than that of the practical diet and could affect normal digestion and absorption, leading to more endogenous energy loss (EEL). Thus, it has been proposed that protein feedstuffs should be fed in combination with a basal diet, whereas cereal grains could be assayed alone (Lockhart et al., 1967; Farrell, 1978; Muztar and Slinger, 1980; Lopez and Leeson, 2008; Zhao et al., 2008). Furthermore, it has been shown that when the test material was mixed with different types of basal diets (Sirbald et al., 1962; Dale and Fuller, 1981; Adeola and Ileleji, 2009) or in different proportions (Leeson et al., 1977), the ME values of the test material varied significantly. It was suggested that there were possible interactions between the test material and the basal diet and that the components of
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RESEARCH NOTE
the basal diet could affect the TME of the test material. Researchers have tried to use glucose as a purified basal diet in the TME assay, but the TME of glucose showed a large range. Moreover, the potential rapid absorption in the duodenum renders glucose unsuitable as a basal diet alone (Anderson et al., 1958; Potter, 1979; Riesenfeld et al., 1980). Many other feedstuffs were also used as basal diets in metabolism trials, such as corn, a practical complete diet and a combination of glucose and starch (Muztar and Slinger, 1980; Lopez and Leeson, 2008; Adeola and Ileleji, 2009). Taking into account the interactions between the test materials and the basal diet, a uniform basal diet should be used to create a reference for test materials. Corn starch was widely used as a purified nitrogen-free basal diet in amino acid utilization assays of protein feedstuffs, which could balance the protein level and also minimize the effect of the components of the basal diet. In this study, we investigated the feasibility of using corn starch as the basal diet to determine the TME of protein feedstuffs so as to provide reliable data for feed formulation.
MATERIALS AND METHODS ME Assay This experiment was conducted at Guangdong Wen’s Foodstuffs Group Co. Ltd. (Guangzhou, China) and was approved by the animal care and handling procedures of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing. The modified TME bioassay described by McNab and Blair (1988) was used in the experiment. The chickens were deprived of feed for 48 h and then fed 40 or 25 g of the test samples via crop intubation. A stainless-steel funnel with a narrow stem of 26 cm long and 8 mm inner diameter was used for force-feeding. Excreta were collected within 48 h according to the method described by Adeola et al. (1997). The EEL was determined in fasted birds during each TME trial. The BW of the chickens were determined before feed withdrawal and after excreta collection. All of the experimental birds were fed the same commercial diet when not under experimental conditions. Water was supplied ad libitum throughout the study via a suspended nipple drinker line. In each TME trail, 18-wk-old Chinese Yellow roosters (Guangxi Yellow chicken 2) were sorted according to their initial weight and placed in individual cages (0.50 × 0.42 × 0.55 m) in an environmentally controlled room (25°C) under 12 h of light per day. All of the birds were healthy throughout the study. After completion of excreta collection in each metabolism trial, all of the samples were dried at 65°C in an oven for 48 h and then equilibrated with air for 24 h. The samples of the excreta and diets were ground in a laboratory mill fitted with a 0.5-mm mesh screen before analysis. The DM contents of the diets and excreta samples were determined by oven drying at 105°C for 5
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h. The energy contents of the diets and excreta samples were determined by bomb calorimetry using a Parr 1281 automatic adiabatic calorimeter (Parr Instrument Co., Moline, IL). The nitrogen determination was according to the combustion method, using an FP2000 nitrogen analyzer (Leco Corp., St. Joseph, MI). The diets were also analyzed for crude fiber (method 962.09) and ether extract (method 920.39) according to the procedures of the AOAC (1990). Each chemical component of the samples was determined in duplicate. The TME of the test diet was calculated according the procedure described by Sirbald (1976).
Experimental Design In experiment 1, the objective was to determine the TME value of corn starch and test the repeatability of the bioassay. In total, 72 roosters were randomly selected according to their initial BW and divided into 3 groups of 24 birds (6 replicates of 4 roosters). One group was randomly chosen to determine the EEL, and the remaining 2 groups were used to determine the ME content of corn starch (0.3% CP) and were tube-fed either 25 or 40 g of corn starch. To test the repeatability of the bioassay, the same experiment was repeated 4 times and 72 different roosters were used in each assay. In experiment 2, the objective was to examine the accuracy of the TME value of individual protein feedstuffs when using corn starch as the basal diet. In total, 168 roosters were randomly divided into 7 groups of 24 birds (6 replicates of 4 roosters) for 7 feeding regimens: fasted, corn, soybean meal (SBM), cottonseed meal (CM), corn-soybean meal diet (CSM), corn-cottonseed meal diet (CCM), and corn-soybean meal-cottonseed meal diet (CSCM). Birds were tubefed 40 g of the test diet, except the fast treatment that was used for determining the EEL. The TME values of the SBM and CM were determined by mixing with the corn starch basal diet in a ratio of 4:6, whereas the corn was fed alone. To test the accuracy of the TME in the corn, SBM, and CM, an additivity examination was conducted to determine the TME values of the CSM, CCM, and CSCM; all of the diets were formulated to contain 17% CP and 4.5 Mcal/kg of gross energy. The composition of the experimental diets and their chemical characteristics are shown in Table 1.
Statistical Analysis The statistical analysis of the data was performed using PROC ANOVA procedures, and the correlation analysis was done using PROC CORR procedures of the SAS version 8 program (SAS Institute, 1990). The treatment mean differences were separated for statistical significance (P < 0.05) by Duncan’s difference test. The t-test was used to measure the significance of the differences between the observed and calculated TME values.
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Table 1. Composition and nutrient content of the experimental diets Diet1 Item Ingredient, % Corn Soybean meal Cottonseed meal Corn starch Premix2 Total Nutrient content DM, % Gross energy, kcal/kg CP, % Crude fiber, % Ether extract, % Calcium,3 % Total P,3 %
Corn
SBM
CM
CSM
CCM
CSCM
99.00 — — — 1.00 100
— 38.92 — 60.08 1.00 100
— — 39.65 59.35 1.00 100
77.30 21.69 — — 1.00 100
78.78 — 20.20 — 1.00 100
77.06 11.80 10.14 — 1.00 100
89.9 4,456 8.39 1.83 4.29 0.02 0.27
88.9 4,735 19.4 1.58 0.91 0.13 0.26
87.2 4,729 21.6 3.82 0.48 0.11 0.42
89.9 4,530 17.3 2.31 2.79 0.09 0.35
87.3 4,529 17.6 3.41 3.6 0.07 0.43
90.6 4,523 17.8 2.88 3.67 0.08 0.39
1SBM = soybean meal; CM = cottonseed meal; CSM = corn-soybean meal diet; CCM = corn-cottonseed meal diet; and CSCM = corn-soybean meal-cottonseed meal diet. 2Supplied per kilogram of diet: vitamin A, 2,700 IU; vitamin D , 400 IU; vitamin E, 10 IU; vitamin K , 0.5 mg; thiamine, 2.0 mg; riboflavin, 5.0 3 3 mg; pantothenic acid, 10.0 mg; niacin, 30 mg; pyridoxine, 3.0 mg; choline, 750 mg; folic acid, 0.5 mg; biotin, 120 µg; vitamin B12, 10 µg; ethoxyquin, 120 mg; Mn, 80 mg; Zn, 80 mg; Cu, 8 mg; Fe, 80 mg; I, 0.7 mg; and Se, 0.3 mg. 3These values were calculated.
RESULTS AND DISCUSSION
assay were significantly lower than those of the others (P < 0.05). A correlation analysis suggested that the EEL has no significant correlation with the initial weight, final weight, weight loss, or endogenous excreta energy per gram, whereas it has a significant positive correction with the excreta weight (P < 0.01; Table 3). It is further suggested that the variation of the EEL was mainly caused by the excreta weight but had no significant relationship with BW. The TME bioassay of the corn starch is shown in Table 4. This bioassay indicated that when the chickens were tube-fed the same level of corn starch (25 or 40 g), the excreta weight, DM metabolizability, energy metabolizability, and TME were similar across all of the assays, showing good reproducibility. With forcefeeding of different levels of corn starch, the TME value of the 25-g treatment (4.06 kcal/g) was significantly higher (P < 0.01) than that of the 40-g treatment (3.79 kcal/g). However, this result was contrary to the theoretical assumptions of the TME bioassay that the TME value of the test ingredient was independent of the feed
In the present study, the assay of the EEL, the endogenous excreta energy per gram, and the excreta weight were uniform across the 4 assays, indicating a good repeatability (Table 2). The mean excreta weight was estimated to be 6.26 g/bird per 48 h, and the EEL was estimated to be 16.76 to 18.46 kcal, with a mean of 17.37 kcal per 48 h for 96 Chinese Yellow chickens. This value was slightly lower than the mean value of 22.42 kcal for 12 Single-Comb White Leghorn roosters in 48 h from the study of Sirbald and Morse (1982). Such a difference may be caused by different species, ages, and collection methods. The original description of the TME bioassay stated that birds used to measure the EEL should be of a similar weight to those fed the test diet (Sirbald, 1976). However, subsequent research showed the absence of a close relationship between the EEL and BW (Farrell, 1978). In our study, the EEL and weight loss were uniform across the 4 assays, though the initial and final weights of the first Table 2. Animal performance and endogenous losses on DM basis Initial weight, kg Assay
Mean
1 (n = 24) 2 (n = 24) 3 (n = 24) 4 (n = 24) Mean SEM P-value a,bMeans 1EEL
2.59b 2.81a 2.84a 2.89a
2.78 0.035 <0.001
Final weight, kg
CV, % 2.21 3.57 2.68 3.46
Mean
2.39b 2.62a 2.69a 2.72a
2.60 0.035 <0.001
Weight loss, kg
CV, % 1.95 4.26 2.75 3.64
Mean
CV, %
0.20 0.19 0.16 0.17 0.18 0.010 0.082
7.78 19.41 8.36 11.76
Endogenous excreta energy, kcal/kg
Excreta, g/48 h
Mean
CV, %
6.09 6.04 6.22 6.70 6.26 0.211 0.139
7.80 5.95 11.67 6.39
in the same column with no common superscript differ significantly (P < 0.05). = endogenous energy loss.
Mean
CV, %
2,753 2,822 2,774 2,755 2,776 24.89 0.215
2.12 3.26 1.30 1.52
EEL,1 kcal/48 h
Mean
CV, %
16.76 17.02 17.24 18.46 17.37 0.538 0.153
8.25 4.11 10.77 5.68
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RESEARCH NOTE Table 3. Analysis of the correlation of endogenous energy loss (EEL, kcal/48 h) with weight and excreta energy on DM basis Correlation coefficient
Assay 1 2 3 4
(n (n (n (n
= = = =
24) 24) 24) 24)
Initial weight, kg
Final weight, kg
Weight loss, kg
Excreta, g/48 h
Endogenous excreta energy, kcal/kg
0.424 −0.188 −0.196 −0.662
0.278 −0.338 −0.298 −0.662
0.523 0.445 0.448 0.08
0.965** 0.841* 0.998** 0.975**
0.287 −0.285 −0.714 −0.357
*The means correlated significantly (P < 0.05). **The means correlated significantly (P < 0.01).
input (Sirbald, 1976). Previous reports have shown that this phenomenon would occur if the excreta collection period was insufficient or the amount fed exceeded the ability of the animals to digest and absorb (Muztar and Slinger, 1979; Sirbald and Morse, 1982). In this study, a 48-h collection time was sufficient, given the fact that corn starch has a high metabolizability and the feed input was 25 or 40 g (McNab and Blair, 1988). In contrast, the DM metabolizability and energy metabolizability values of the 25-g treatment (101.25 and 98.06%, respectively) were significantly higher (P < 0.01) than those of the 40-g treatment (91.59 and 94.52%, respectively). So it could be inferred that force-feeding 40 g of corn starch (as a purified ingredient) in one minute would exceed the normal metabolizability of Chinese Yellow chickens. Therefore, it is reasonable to use the TME of the 25-g treatment in feed formulation. In addition, the TME of corn starch in the 25-g treatment was equal to 98.06% of its energy content, indicating a nearly complete absorption. Taking into account that the ratio of calories to protein in the mixed diet is more balanced than individual ingredients, the maximal increase in utilization was 1.96% (98.06–100%), which was within the acceptable range of error for animal experiments. The CV of the TME between the 4 assays of the 25-g treatment was 1.60%, indicating a satisfactory
precision. Therefore, it was suitable to use corn starch as the basal diet in determining the TME value of the protein feedstuff. Soybean meal and CM are commonly used as protein feedstuffs for Chinese Yellow chickens, and their TME values were determined using corn starch as the basal diet in the present study. When designing experiments to measure the energy metabolizability of protein feedstuffs, it is often difficult to determine what level of protein feedstuffs should be included in the mixed diet. A small quantity of protein feedstuffs results in a greater chance of having mixed diets and practical diets of similar nutrient balances, but a lower level of protein feedstuffs results in greater magnification of small experimental errors (Lopez and Leeson, 2008). Therefore, many researchers have suggested that the proportion of protein feedstuffs should be between 30 to 50% (Farrell, 1978; Mohamed et al., 1984; Farrell, 1999). In our study, when the corn starch was mixed with the SBM (48.99% CP) or CM (53.35% CP) in a ratio of 60:40, the protein levels of the mixed diets were approximately 20% and were similar to that in practical diets. When the amount fed to the chickens in the TME assay was 40 g, the actual intake of corn starch was approximately 24 g and was within the normal digestion limits of chickens. According to the TME value of corn starch obtained
Table 4. The effect of amount fed on excreta excretion, true ME, and energy metabolizability of corn starch on DM basis DM metabolizability, %
Excreta, g/48 h Corn starch, g
Assay
Mean
CV, %
40
1 (n = 24) 2 (n = 24) 3 (n = 24) 4 (n = 24) SEM P-value 1 (n = 24) 2 (n = 24) 3 (n = 24) 4 (n = 24) SEM P-value 4 assays 4 assays SEM P-value
9.10 8.04 7.85 7.84 0.654 0.482 5.74 6.06 5.97 6.07 0.189 0.613 8.21 5.97 0.247 <0.001
22.42 26.66 10.60 11.60 5.55 7.16 5.41 9.78 19.34 7.21
25
40 25
Mean
CV, %
91.95 94.94 95.57 95.61 1.852 0.467 102.2 100.9 101.2 100.8 0.838 0.678 94.52 101.3 0.737 <0.001
6.28 6.43 2.43 2.64 1.31 1.91 1.35 2.66 4.76 1.87
Energy metabolizability, %
True ME, kcal/g
Mean
CV, %
3.69 3.79 3.84 3.83 0.073 0.470 4.09 4.02 4.09 4.06 0.026 0.186 3.79 4.06 0.028 <0.001
6.24 6.30 1.98 2.69 0.92 2.15 1.35 1.28 4.65 1.60
Mean
CV, %
89.16 91.39 92.72 92.54 1.752 0.471 98.77 96.97 98.73 97.99 0.638 0.187 91.45 98.06 0.681 <0.001
6.24 6.30 1.98 2.69 0.92 2.15 1.35 1.28 4.65 1.60
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Ren et al. Table 5. Degree of additivity of the true ME values on DM basis True ME, kcal/g Diet1 Corn SM CM CSM CCM CSCM
Determined (± SD) 4.02 3.39 2.92 3.89 3.77 3.82
± ± ± ± ± ±
0.04 0.10 0.12 0.05 0.05 0.03
Calculated
SEM
P-value
Deviation,2 %
— — — 3.88 3.79 3.83
— — — 0.061 0.052 0.036
— — — 0.814 0.693 0.825
— — — 0.257 −0.531 −0.262
1SM = soybean meal; CM = cottonseed meal; CSM = corn-soybean meal diet; CCM = corn-cottonseed meal diet; and CSCM = corn-soybean meal-cottonseed meal diet. 2Deviation was calculated as (determined − calculated)/determined × 100%.
from the 25-g treatment and the relative proportions of corn starch in the mixed diets, the TME content of SBM and CM were then calculated and shown in Table 5. For corn, as a cereal feedstuff, the TME was assayed when it was provided alone. It was noted that corn had a TME of 4.02 ± 0.04 kcal/g (Table 5) and was similar to the values reported by Sirbald (1976), Muztar and Slinger (1981), and Luis and Sullivan (1982). Soybean meal containing 48.99% CP yielded 3.39 ± 0.10 kcal/g of TME in the present study, which was higher than 2.91 to 3.28 kcal/g of TME of SBM with 49% CP reported by Dale and Fuller (1980), Muztar and Slinger (1980), and Kessler and Thomas(1981) and was also higher than 2.80 kcal/g of TME of SBM with 45% CP reported by Muztar and Slinger (1981). Cottonseed meal containing 53.35% CP had a TME of 2.92 ± 0.12 kcal/g in our study, whereas Dale and Fuller (1984) and Nadeem et al. (2005) noted lower TME values (2.46 and 2.52 kcal/g, respectively) of CM containing 40.90 and 49.04% CP, respectively. It was indicated that the TME values of SBM and CM in our study were higher than in previous studies in which the meals were fed alone. Although the age and species of the animals and the protein feedstuffs used in the previously mentioned studies may have contributed to the variation in the measurements, the TME bioassay method, such as the excreta collection time and the use of corn starch as the basal diet, may primarily account for the differences between our study and the aforementioned reports. A fundamental assumption in poultry feed formulation is that the nutrient supply of individual feedstuffs can be added together to meet the nutrient specifications of the diet, but this has rarely been tested experimentally. In the present study, the additivity examination was performed to determine the TME values of the CSM, CCM, and CSCM. The differences between the observed TME values and the predicted values from measurement that was determined with individual ingredients are shown in Table 5. The observed and predicted TME values of CSM, CCM, and CSCM were in high agreement, with differences ranging from −0.02 to 0.01 kcal/g, and none of the differences were significant, indicating that the TME of corn, SBM, and CM were all additive and did not have any significant associative effects in chickens. Sirbald (1976) observed
a 2.0% deviation in TME in a mixed diet of CSM between the determined and calculated values in roosters, whereas the corresponding values for 3 mixed diets of corn, SBM, and either poultry by-product meal, corngluten meal, or fish meal were 0.56 to 6.7% in broilers (Dale and Fuller, 1980) and 2 mixed diets of corn, SBM, and wheat red dog were 2.1 and 2.4% in ducks (Hong et al., 2002), among which all of the individual feedstuffs were determined by feeding alone. In the present study, the maximum deviation of the TME in the 3 diet mixtures between the calculations and the determined values was close to 0.5%, indicating an accurate measurement of the ME of the individual ingredients using corn starch as the basal diet. Besides, the TME value of corn starch was found to be close to that of corn (4.06 vs. 4.02 kcal/g). So, when the ratio of corn starch and protein feedstuff fluctuates between 60:40 to approximately 70:30, not only the protein content but also the TME of mixed diets were similar to practical diets. Accordingly the measurements of protein feedstuffs were closer to the actual values in practices of commercial husbandry. The formulation of chicken diets should rely upon accurate information regarding the bioavailable energy content of the feedstuffs. In this study, corn starch, as a purified ingredient and with a high energy metabolizability, was used as a basal diet in determining the TME of protein feedstuffs, and the TME values of the test ingredients indicated a satisfactory additivity. Because the ultimate absorption of corn starch cannot be affected by the supplementation of feedstuffs and because being free of nitrogen can balance the protein content in a mixed diet, the use of corn starch provides a valid control for measuring high-protein feedstuffs in TME bioassays and enables both energy and amino acid utilization assays to be efficiently performed in the same experiment. Notably, this study has only examined 2 common protein feedstuffs using corn starch as the basal diet, and additional further research is required.
ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (30901037), State Innovation
RESEARCH NOTE
Method Project (2009IM033100) and Wen’s Foodstuffs Group (Guangzhou, China). We also thank S. J. Liu, Y. G. Yin, Z. K. Liu, J. Z. Tan, and B. M. Mi (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing) for their help in force-feeding and excreta sample collection.
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between the 4 assays were noted. The TME and energy metabolizability of the 25-g corn starch treatment (4.06 kcal/g and 98.06%) were higher than those of the 40-g treatment (3.79 kcal/g and 91.45%; P < 0.01); whereas the CV were less than that of the 40-g treatment, indicating that it is reasonable to use the TME value of the 25-g treatment in feed formulation. In experiment 2, the TME values for corn, soybean meal, and cottonseed meal were 4.02, 3.39, and 2.92 kcal/g, respectively. The observed and predicted TME values of the corn-soybean meal, corn-cottonseed meal, and corn-soybean meal-cottonseed meal diets were in high agreement with differences ranging from −0.02 to 0.01 kcal/g. None of the differences was significant, indicating an accurate measure of the TME of the individual ingredients. Thus, using corn starch as the basal diet to determine the TME of protein feedstuffs was validated.
Key words: basal diet, Chinese Yellow chicken, corn starch, protein feedstuff, true metabolizable energy 2012 Poultry Science 91:1394–1399 http://dx.doi.org/10.3382/ps.2011-01838
INTRODUCTION The Chinese Yellow chicken is a crossbreed of the recessive white hen and the Chinese-native Yellow rooster, and it is characterized by slow growth, light market weight, and good taste and meat quality. In the recent 5 yr, the annual total Chinese Yellow chickens were about 3 billion, which accounted for one-half of China’s total production of broilers (NPIA-CAAA, 2008). However, data are still scarce on the utilization of nutrients in feedstuffs for the Chinese Yellow chickens. Metabolizable energy is an important dietary energy utilization response criterion in feed formulation and represents a large portion of the total cost of the broiler industry. So, it is especially critical to select a precise and accurate method to determine the ME of feedstuffs in Chinese Yellow chickens. ©2012 Poultry Science Association Inc. Received September 1, 2011. Accepted November 19, 2011. 1 Corresponding author: [email protected]
Developed by Sirbald (1976), the accepted procedure for measuring the TME of feedstuffs has been widely used in more than 30 countries (Dudley-Cash, 2009). This method enables the single feedstuff to be fed in a precisely measured quantity, which is carried out rapidly and inexpensively. However, it was found that when protein feedstuff was tube-fed alone, the protein content was much higher than that of the practical diet and could affect normal digestion and absorption, leading to more endogenous energy loss (EEL). Thus, it has been proposed that protein feedstuffs should be fed in combination with a basal diet, whereas cereal grains could be assayed alone (Lockhart et al., 1967; Farrell, 1978; Muztar and Slinger, 1980; Lopez and Leeson, 2008; Zhao et al., 2008). Furthermore, it has been shown that when the test material was mixed with different types of basal diets (Sirbald et al., 1962; Dale and Fuller, 1981; Adeola and Ileleji, 2009) or in different proportions (Leeson et al., 1977), the ME values of the test material varied significantly. It was suggested that there were possible interactions between the test material and the basal diet and that the components of
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RESEARCH NOTE
the basal diet could affect the TME of the test material. Researchers have tried to use glucose as a purified basal diet in the TME assay, but the TME of glucose showed a large range. Moreover, the potential rapid absorption in the duodenum renders glucose unsuitable as a basal diet alone (Anderson et al., 1958; Potter, 1979; Riesenfeld et al., 1980). Many other feedstuffs were also used as basal diets in metabolism trials, such as corn, a practical complete diet and a combination of glucose and starch (Muztar and Slinger, 1980; Lopez and Leeson, 2008; Adeola and Ileleji, 2009). Taking into account the interactions between the test materials and the basal diet, a uniform basal diet should be used to create a reference for test materials. Corn starch was widely used as a purified nitrogen-free basal diet in amino acid utilization assays of protein feedstuffs, which could balance the protein level and also minimize the effect of the components of the basal diet. In this study, we investigated the feasibility of using corn starch as the basal diet to determine the TME of protein feedstuffs so as to provide reliable data for feed formulation.
MATERIALS AND METHODS ME Assay This experiment was conducted at Guangdong Wen’s Foodstuffs Group Co. Ltd. (Guangzhou, China) and was approved by the animal care and handling procedures of the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing. The modified TME bioassay described by McNab and Blair (1988) was used in the experiment. The chickens were deprived of feed for 48 h and then fed 40 or 25 g of the test samples via crop intubation. A stainless-steel funnel with a narrow stem of 26 cm long and 8 mm inner diameter was used for force-feeding. Excreta were collected within 48 h according to the method described by Adeola et al. (1997). The EEL was determined in fasted birds during each TME trial. The BW of the chickens were determined before feed withdrawal and after excreta collection. All of the experimental birds were fed the same commercial diet when not under experimental conditions. Water was supplied ad libitum throughout the study via a suspended nipple drinker line. In each TME trail, 18-wk-old Chinese Yellow roosters (Guangxi Yellow chicken 2) were sorted according to their initial weight and placed in individual cages (0.50 × 0.42 × 0.55 m) in an environmentally controlled room (25°C) under 12 h of light per day. All of the birds were healthy throughout the study. After completion of excreta collection in each metabolism trial, all of the samples were dried at 65°C in an oven for 48 h and then equilibrated with air for 24 h. The samples of the excreta and diets were ground in a laboratory mill fitted with a 0.5-mm mesh screen before analysis. The DM contents of the diets and excreta samples were determined by oven drying at 105°C for 5
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h. The energy contents of the diets and excreta samples were determined by bomb calorimetry using a Parr 1281 automatic adiabatic calorimeter (Parr Instrument Co., Moline, IL). The nitrogen determination was according to the combustion method, using an FP2000 nitrogen analyzer (Leco Corp., St. Joseph, MI). The diets were also analyzed for crude fiber (method 962.09) and ether extract (method 920.39) according to the procedures of the AOAC (1990). Each chemical component of the samples was determined in duplicate. The TME of the test diet was calculated according the procedure described by Sirbald (1976).
Experimental Design In experiment 1, the objective was to determine the TME value of corn starch and test the repeatability of the bioassay. In total, 72 roosters were randomly selected according to their initial BW and divided into 3 groups of 24 birds (6 replicates of 4 roosters). One group was randomly chosen to determine the EEL, and the remaining 2 groups were used to determine the ME content of corn starch (0.3% CP) and were tube-fed either 25 or 40 g of corn starch. To test the repeatability of the bioassay, the same experiment was repeated 4 times and 72 different roosters were used in each assay. In experiment 2, the objective was to examine the accuracy of the TME value of individual protein feedstuffs when using corn starch as the basal diet. In total, 168 roosters were randomly divided into 7 groups of 24 birds (6 replicates of 4 roosters) for 7 feeding regimens: fasted, corn, soybean meal (SBM), cottonseed meal (CM), corn-soybean meal diet (CSM), corn-cottonseed meal diet (CCM), and corn-soybean meal-cottonseed meal diet (CSCM). Birds were tubefed 40 g of the test diet, except the fast treatment that was used for determining the EEL. The TME values of the SBM and CM were determined by mixing with the corn starch basal diet in a ratio of 4:6, whereas the corn was fed alone. To test the accuracy of the TME in the corn, SBM, and CM, an additivity examination was conducted to determine the TME values of the CSM, CCM, and CSCM; all of the diets were formulated to contain 17% CP and 4.5 Mcal/kg of gross energy. The composition of the experimental diets and their chemical characteristics are shown in Table 1.
Statistical Analysis The statistical analysis of the data was performed using PROC ANOVA procedures, and the correlation analysis was done using PROC CORR procedures of the SAS version 8 program (SAS Institute, 1990). The treatment mean differences were separated for statistical significance (P < 0.05) by Duncan’s difference test. The t-test was used to measure the significance of the differences between the observed and calculated TME values.
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Table 1. Composition and nutrient content of the experimental diets Diet1 Item Ingredient, % Corn Soybean meal Cottonseed meal Corn starch Premix2 Total Nutrient content DM, % Gross energy, kcal/kg CP, % Crude fiber, % Ether extract, % Calcium,3 % Total P,3 %
Corn
SBM
CM
CSM
CCM
CSCM
99.00 — — — 1.00 100
— 38.92 — 60.08 1.00 100
— — 39.65 59.35 1.00 100
77.30 21.69 — — 1.00 100
78.78 — 20.20 — 1.00 100
77.06 11.80 10.14 — 1.00 100
89.9 4,456 8.39 1.83 4.29 0.02 0.27
88.9 4,735 19.4 1.58 0.91 0.13 0.26
87.2 4,729 21.6 3.82 0.48 0.11 0.42
89.9 4,530 17.3 2.31 2.79 0.09 0.35
87.3 4,529 17.6 3.41 3.6 0.07 0.43
90.6 4,523 17.8 2.88 3.67 0.08 0.39
1SBM = soybean meal; CM = cottonseed meal; CSM = corn-soybean meal diet; CCM = corn-cottonseed meal diet; and CSCM = corn-soybean meal-cottonseed meal diet. 2Supplied per kilogram of diet: vitamin A, 2,700 IU; vitamin D , 400 IU; vitamin E, 10 IU; vitamin K , 0.5 mg; thiamine, 2.0 mg; riboflavin, 5.0 3 3 mg; pantothenic acid, 10.0 mg; niacin, 30 mg; pyridoxine, 3.0 mg; choline, 750 mg; folic acid, 0.5 mg; biotin, 120 µg; vitamin B12, 10 µg; ethoxyquin, 120 mg; Mn, 80 mg; Zn, 80 mg; Cu, 8 mg; Fe, 80 mg; I, 0.7 mg; and Se, 0.3 mg. 3These values were calculated.
RESULTS AND DISCUSSION
assay were significantly lower than those of the others (P < 0.05). A correlation analysis suggested that the EEL has no significant correlation with the initial weight, final weight, weight loss, or endogenous excreta energy per gram, whereas it has a significant positive correction with the excreta weight (P < 0.01; Table 3). It is further suggested that the variation of the EEL was mainly caused by the excreta weight but had no significant relationship with BW. The TME bioassay of the corn starch is shown in Table 4. This bioassay indicated that when the chickens were tube-fed the same level of corn starch (25 or 40 g), the excreta weight, DM metabolizability, energy metabolizability, and TME were similar across all of the assays, showing good reproducibility. With forcefeeding of different levels of corn starch, the TME value of the 25-g treatment (4.06 kcal/g) was significantly higher (P < 0.01) than that of the 40-g treatment (3.79 kcal/g). However, this result was contrary to the theoretical assumptions of the TME bioassay that the TME value of the test ingredient was independent of the feed
In the present study, the assay of the EEL, the endogenous excreta energy per gram, and the excreta weight were uniform across the 4 assays, indicating a good repeatability (Table 2). The mean excreta weight was estimated to be 6.26 g/bird per 48 h, and the EEL was estimated to be 16.76 to 18.46 kcal, with a mean of 17.37 kcal per 48 h for 96 Chinese Yellow chickens. This value was slightly lower than the mean value of 22.42 kcal for 12 Single-Comb White Leghorn roosters in 48 h from the study of Sirbald and Morse (1982). Such a difference may be caused by different species, ages, and collection methods. The original description of the TME bioassay stated that birds used to measure the EEL should be of a similar weight to those fed the test diet (Sirbald, 1976). However, subsequent research showed the absence of a close relationship between the EEL and BW (Farrell, 1978). In our study, the EEL and weight loss were uniform across the 4 assays, though the initial and final weights of the first Table 2. Animal performance and endogenous losses on DM basis Initial weight, kg Assay
Mean
1 (n = 24) 2 (n = 24) 3 (n = 24) 4 (n = 24) Mean SEM P-value a,bMeans 1EEL
2.59b 2.81a 2.84a 2.89a
2.78 0.035 <0.001
Final weight, kg
CV, % 2.21 3.57 2.68 3.46
Mean
2.39b 2.62a 2.69a 2.72a
2.60 0.035 <0.001
Weight loss, kg
CV, % 1.95 4.26 2.75 3.64
Mean
CV, %
0.20 0.19 0.16 0.17 0.18 0.010 0.082
7.78 19.41 8.36 11.76
Endogenous excreta energy, kcal/kg
Excreta, g/48 h
Mean
CV, %
6.09 6.04 6.22 6.70 6.26 0.211 0.139
7.80 5.95 11.67 6.39
in the same column with no common superscript differ significantly (P < 0.05). = endogenous energy loss.
Mean
CV, %
2,753 2,822 2,774 2,755 2,776 24.89 0.215
2.12 3.26 1.30 1.52
EEL,1 kcal/48 h
Mean
CV, %
16.76 17.02 17.24 18.46 17.37 0.538 0.153
8.25 4.11 10.77 5.68
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RESEARCH NOTE Table 3. Analysis of the correlation of endogenous energy loss (EEL, kcal/48 h) with weight and excreta energy on DM basis Correlation coefficient
Assay 1 2 3 4
(n (n (n (n
= = = =
24) 24) 24) 24)
Initial weight, kg
Final weight, kg
Weight loss, kg
Excreta, g/48 h
Endogenous excreta energy, kcal/kg
0.424 −0.188 −0.196 −0.662
0.278 −0.338 −0.298 −0.662
0.523 0.445 0.448 0.08
0.965** 0.841* 0.998** 0.975**
0.287 −0.285 −0.714 −0.357
*The means correlated significantly (P < 0.05). **The means correlated significantly (P < 0.01).
input (Sirbald, 1976). Previous reports have shown that this phenomenon would occur if the excreta collection period was insufficient or the amount fed exceeded the ability of the animals to digest and absorb (Muztar and Slinger, 1979; Sirbald and Morse, 1982). In this study, a 48-h collection time was sufficient, given the fact that corn starch has a high metabolizability and the feed input was 25 or 40 g (McNab and Blair, 1988). In contrast, the DM metabolizability and energy metabolizability values of the 25-g treatment (101.25 and 98.06%, respectively) were significantly higher (P < 0.01) than those of the 40-g treatment (91.59 and 94.52%, respectively). So it could be inferred that force-feeding 40 g of corn starch (as a purified ingredient) in one minute would exceed the normal metabolizability of Chinese Yellow chickens. Therefore, it is reasonable to use the TME of the 25-g treatment in feed formulation. In addition, the TME of corn starch in the 25-g treatment was equal to 98.06% of its energy content, indicating a nearly complete absorption. Taking into account that the ratio of calories to protein in the mixed diet is more balanced than individual ingredients, the maximal increase in utilization was 1.96% (98.06–100%), which was within the acceptable range of error for animal experiments. The CV of the TME between the 4 assays of the 25-g treatment was 1.60%, indicating a satisfactory
precision. Therefore, it was suitable to use corn starch as the basal diet in determining the TME value of the protein feedstuff. Soybean meal and CM are commonly used as protein feedstuffs for Chinese Yellow chickens, and their TME values were determined using corn starch as the basal diet in the present study. When designing experiments to measure the energy metabolizability of protein feedstuffs, it is often difficult to determine what level of protein feedstuffs should be included in the mixed diet. A small quantity of protein feedstuffs results in a greater chance of having mixed diets and practical diets of similar nutrient balances, but a lower level of protein feedstuffs results in greater magnification of small experimental errors (Lopez and Leeson, 2008). Therefore, many researchers have suggested that the proportion of protein feedstuffs should be between 30 to 50% (Farrell, 1978; Mohamed et al., 1984; Farrell, 1999). In our study, when the corn starch was mixed with the SBM (48.99% CP) or CM (53.35% CP) in a ratio of 60:40, the protein levels of the mixed diets were approximately 20% and were similar to that in practical diets. When the amount fed to the chickens in the TME assay was 40 g, the actual intake of corn starch was approximately 24 g and was within the normal digestion limits of chickens. According to the TME value of corn starch obtained
Table 4. The effect of amount fed on excreta excretion, true ME, and energy metabolizability of corn starch on DM basis DM metabolizability, %
Excreta, g/48 h Corn starch, g
Assay
Mean
CV, %
40
1 (n = 24) 2 (n = 24) 3 (n = 24) 4 (n = 24) SEM P-value 1 (n = 24) 2 (n = 24) 3 (n = 24) 4 (n = 24) SEM P-value 4 assays 4 assays SEM P-value
9.10 8.04 7.85 7.84 0.654 0.482 5.74 6.06 5.97 6.07 0.189 0.613 8.21 5.97 0.247 <0.001
22.42 26.66 10.60 11.60 5.55 7.16 5.41 9.78 19.34 7.21
25
40 25
Mean
CV, %
91.95 94.94 95.57 95.61 1.852 0.467 102.2 100.9 101.2 100.8 0.838 0.678 94.52 101.3 0.737 <0.001
6.28 6.43 2.43 2.64 1.31 1.91 1.35 2.66 4.76 1.87
Energy metabolizability, %
True ME, kcal/g
Mean
CV, %
3.69 3.79 3.84 3.83 0.073 0.470 4.09 4.02 4.09 4.06 0.026 0.186 3.79 4.06 0.028 <0.001
6.24 6.30 1.98 2.69 0.92 2.15 1.35 1.28 4.65 1.60
Mean
CV, %
89.16 91.39 92.72 92.54 1.752 0.471 98.77 96.97 98.73 97.99 0.638 0.187 91.45 98.06 0.681 <0.001
6.24 6.30 1.98 2.69 0.92 2.15 1.35 1.28 4.65 1.60
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Ren et al. Table 5. Degree of additivity of the true ME values on DM basis True ME, kcal/g Diet1 Corn SM CM CSM CCM CSCM
Determined (± SD) 4.02 3.39 2.92 3.89 3.77 3.82
± ± ± ± ± ±
0.04 0.10 0.12 0.05 0.05 0.03
Calculated
SEM
P-value
Deviation,2 %
— — — 3.88 3.79 3.83
— — — 0.061 0.052 0.036
— — — 0.814 0.693 0.825
— — — 0.257 −0.531 −0.262
1SM = soybean meal; CM = cottonseed meal; CSM = corn-soybean meal diet; CCM = corn-cottonseed meal diet; and CSCM = corn-soybean meal-cottonseed meal diet. 2Deviation was calculated as (determined − calculated)/determined × 100%.
from the 25-g treatment and the relative proportions of corn starch in the mixed diets, the TME content of SBM and CM were then calculated and shown in Table 5. For corn, as a cereal feedstuff, the TME was assayed when it was provided alone. It was noted that corn had a TME of 4.02 ± 0.04 kcal/g (Table 5) and was similar to the values reported by Sirbald (1976), Muztar and Slinger (1981), and Luis and Sullivan (1982). Soybean meal containing 48.99% CP yielded 3.39 ± 0.10 kcal/g of TME in the present study, which was higher than 2.91 to 3.28 kcal/g of TME of SBM with 49% CP reported by Dale and Fuller (1980), Muztar and Slinger (1980), and Kessler and Thomas(1981) and was also higher than 2.80 kcal/g of TME of SBM with 45% CP reported by Muztar and Slinger (1981). Cottonseed meal containing 53.35% CP had a TME of 2.92 ± 0.12 kcal/g in our study, whereas Dale and Fuller (1984) and Nadeem et al. (2005) noted lower TME values (2.46 and 2.52 kcal/g, respectively) of CM containing 40.90 and 49.04% CP, respectively. It was indicated that the TME values of SBM and CM in our study were higher than in previous studies in which the meals were fed alone. Although the age and species of the animals and the protein feedstuffs used in the previously mentioned studies may have contributed to the variation in the measurements, the TME bioassay method, such as the excreta collection time and the use of corn starch as the basal diet, may primarily account for the differences between our study and the aforementioned reports. A fundamental assumption in poultry feed formulation is that the nutrient supply of individual feedstuffs can be added together to meet the nutrient specifications of the diet, but this has rarely been tested experimentally. In the present study, the additivity examination was performed to determine the TME values of the CSM, CCM, and CSCM. The differences between the observed TME values and the predicted values from measurement that was determined with individual ingredients are shown in Table 5. The observed and predicted TME values of CSM, CCM, and CSCM were in high agreement, with differences ranging from −0.02 to 0.01 kcal/g, and none of the differences were significant, indicating that the TME of corn, SBM, and CM were all additive and did not have any significant associative effects in chickens. Sirbald (1976) observed
a 2.0% deviation in TME in a mixed diet of CSM between the determined and calculated values in roosters, whereas the corresponding values for 3 mixed diets of corn, SBM, and either poultry by-product meal, corngluten meal, or fish meal were 0.56 to 6.7% in broilers (Dale and Fuller, 1980) and 2 mixed diets of corn, SBM, and wheat red dog were 2.1 and 2.4% in ducks (Hong et al., 2002), among which all of the individual feedstuffs were determined by feeding alone. In the present study, the maximum deviation of the TME in the 3 diet mixtures between the calculations and the determined values was close to 0.5%, indicating an accurate measurement of the ME of the individual ingredients using corn starch as the basal diet. Besides, the TME value of corn starch was found to be close to that of corn (4.06 vs. 4.02 kcal/g). So, when the ratio of corn starch and protein feedstuff fluctuates between 60:40 to approximately 70:30, not only the protein content but also the TME of mixed diets were similar to practical diets. Accordingly the measurements of protein feedstuffs were closer to the actual values in practices of commercial husbandry. The formulation of chicken diets should rely upon accurate information regarding the bioavailable energy content of the feedstuffs. In this study, corn starch, as a purified ingredient and with a high energy metabolizability, was used as a basal diet in determining the TME of protein feedstuffs, and the TME values of the test ingredients indicated a satisfactory additivity. Because the ultimate absorption of corn starch cannot be affected by the supplementation of feedstuffs and because being free of nitrogen can balance the protein content in a mixed diet, the use of corn starch provides a valid control for measuring high-protein feedstuffs in TME bioassays and enables both energy and amino acid utilization assays to be efficiently performed in the same experiment. Notably, this study has only examined 2 common protein feedstuffs using corn starch as the basal diet, and additional further research is required.
ACKNOWLEDGMENTS The authors thank the National Natural Science Foundation of China (30901037), State Innovation
RESEARCH NOTE
Method Project (2009IM033100) and Wen’s Foodstuffs Group (Guangzhou, China). We also thank S. J. Liu, Y. G. Yin, Z. K. Liu, J. Z. Tan, and B. M. Mi (Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing) for their help in force-feeding and excreta sample collection.
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