| Formulating diets on the true digestible amino acid(TDAA) nutrition system is one of the effective tools for improving amino acid nutrition and reducing N pollution and waste of resources. The technical barrier of establishing TDAA nutrition system is the difficulty in accurate estimation of endogenous amino acids losses(EAAL) of poultry fed actual protein-containing diets. The single-administration of isotope-leucine method, which was expected to be relatively ideal for determining EAAL of poultry, was initially proposed and confirmed by our team with support from National Natural Science Foundation. Howere, further work needs to be done to clarify the reliability of the method within a wide range of crude protein(CP) in diets. The study was conducted to verify the reliability and feasibility of this method in poultry fed actual protein-containing diets ranged from 0 to 21% CP based on isotopic tracer technique with 15N-Leu as tracer.1. The 15N-Leucine Single-Administration Method for Measuring Endogenous Amino Acid Losses in Chicken: Optimization of Main ParametersThe two experiment were designed to optimize parameters of the 15N-leucine single-administration method for measuring endogenous amino acid losses in chicken. Experiment 1 was conducted to provide suitable parameters of gas chromatography-mass spectrometry(GC-MS) for determing 15N-enrichment of leucine(APEL) and ascertain an appropriate scope of APEL in samples. Experiment 2 was carried out to optimize administration parameters of 15N-leucine, using 48 Avian roosters to study the effects of administration methods(intramuscular injection and intravenous injection) and doses(10, 20 and 30 mg/kg·BW) of 15N-leucine on 15N-enrichment in main secretory sources of endogenous amino acids. The results showed as follows:(1) the biggest characteristic fragment ion peaks of MTBSTFA derivatives of leucine in GC-MS is m/z=200. Precision and accuracy of measurement were poor when APEL<0.1% in samples; measurement precision and accuracy respectively ranged from 4.3% to 9.7% and-5.9% to 4.1% when APEL ranged from 0.1% to 3.2%, and the standard curve of mixtures of natural and 15N-leucine showed agood linear relationship(R2=0.989).(2) The values of APEL in main secretory sources of endogenous amino acids were greater than 0.1% for 15N-leucine administration doses of 20 and 30 mg/kg·BW. The experimental birds administrated 15N-leucine through intravenous injection showed harmful stress including congestion and extravasation at the injection sites.(3) There were not administration methods and doses interaction effects(P>0.05) of APEL in main secretory sources of endogenous amino acids. The values of APEL in main secretory sources of endogenous amino acids were increased significantly(P<0.05) with an increase of administration dose. The values of APEL among different secretory sources had no significant difference(P>0.05) at 15N-leucine administration doses of 20 mg/kg·BW. Howere, the values of APEL in pancreas and intestinal mucosa were influenced significantly(P<0.05) by 15N-leucine administration method. In conclusion, the 15N-leucine administration dose of 20 mg/kg·BW is selected to meet the requirements of GC-MS analysis, and intramuscular injection of 15N-leucine is applied to reduce harmful stress, simplify the operating procedure and reduce the cost.2. Evaluation of the 15N-Leucine single-administration method for determing endogenous amino acid losses in cecectomized roosters: effect of dietary crude protein content and the reliability in methodologyThe objective of this experiment was to verify the reliability of the 15N-leucine single-administration method for determing endogenous amino acid losses in poultry fed actual protein-containing diets ranged from 0 to 21% CP. A total of 48 cecectomized roosters were selected and grouped into 8 treaments in a completely randomized design. Each treatment consisted of 6 replicates accommodating one bird per replication. The experimental diets were protein-containing diets(soybean meal served as the sole source of dietary protein) containing crude protein(CP) of 0, 3.0%, 6.0%, 9.0%, 12.0%, 15.0%, 18.0% and 21.0%, respectively. Experimental procedure was performed as Sibbald gavage method, and each bird was precisely fed with experimental diet of 25 g/kg·BW. After the feeding, birds were administrated 15N-leucine by a single intramuscular injection at a rate of 20 mg/kg·BW. Blood samples were taken at 24 h and excreta samples were continuously collected during the course of the 48.0-h experiment after injection. The results showed as follows: 1) The ratio(15NLcm/15NLp) of 15N-enrichment(atom % excess, APE) of leucine in crude mucin(15NLcm) to in the TCA-soluble fraction of plasma(15NLp), which ranged from 0.664 to 0.763, remained relatively consistent(P>0.05) among all treatments. 2) The profile of endogenous amino acids calculated by the graded protein calculation method, expressed as individual amino acid proportion of total amino acids, indicated that the ratios of most amino acids were not influenced(P>0.05) by dietary CP levels with exception of isoleucine, alanine, asparticacid, cysteine, proline and serine. The predominant endogenous amino acids found in the excreta collected form all treatments were glutamic acid, aspartic acid, threonine, serine, proline and valine. The order of the relative proportion of these predominant amino acids also remained relatively consistent(P>0.05) among all treatments. 3) The endogenous losses of total amino acids determined by the 15N-leucine single-administration method were 10.21, 10.96, 13.18, 17.34, 20.51, 20.11, 21.90 and 22.46 g/kg·DMI for different treaments, respectively. The endogenous losses of total amino acid, which were determined by the 15N-leucine single-administration method in birds fed diets containing low and adjacent high CP levels, were not significantly(P>0.05) different from the average endogenous losses of total amino acid calculated by the graded protein calculation method within the range of the low and adjacent high CP levels. In conclusion, this experiment verified the hypothesis of the 15N-leucine single-administration method.In summary, the 15N-leucine single-administration method can be used to measure endogenous amino acid losses in poultry fed actual protein-containing diets within the range of the wider dietary CP levels(0~21%). In addition, our findings suggested that the 15N-leucine dose of 20 mg/kg·BW and intramuscular administration should be selected to applied to actual application. |
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