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* Institute of Food, Nutrition and Human Health, Massey University, Palmerston North, New Zealand and
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Department of Nutritional Physiology "Oskar Kellner" ResearchInstitute for the Biology of Farm Animals, Dummerstorf, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Endogenous Protein • Lysine • Nitrogen • Pigs • Rats
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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; Boisen and Moughan, 1996a). The enzyme-hydrolyzed casein (EHC), isotope dilution (ID), and guanidination (G) methods have been developed to this end.
The EHC method (Moughan et al., 1990) involves feeding animals a diet containing enzymatically hydrolyzed protein (usually casein; molecular weight < 5,000 Da) as the sole N source. Collected digesta are centrifuged and ultrafiltered. The high-molecular weight fraction (>10,000 Da), containing endogenous protein, is analyzed for total N and AA. Unabsorbed dietary AA and N are discarded in the ultrafiltrate.
The ID method can be applied by feeding diets labeled with 15N to animals (15N-dilution method; Leterme et al., 1993; Roos et al., 1994) or by labeling the animal’s body N-pool using 15N-leucine (15N-infusion method; de Lange et al., 1990; Huisman et al., 1992). Endogenous N (EN) in the digesta can be distinguished from exogenous (dietary) N by reference to the isotope enrichment of the digesta.
The G method involves partially transforming dietary Lys to homoarginine, which is absorbed but is not present in endogenous gut protein, thus acting as a marker for Lys uptake. By determining both the homoarginine and Lys contents of ileal digesta collected after feeding an animal a guanidinated diet, the endogenous lysine (EL) flow can be determined (Hagemeister and Erbersdobler, 1985; Rutherfurd and Moughan, 1990).
The EHC method allows a direct estimation of both EN and EL, whereas the ID method and the G method only allow direct determination of EN and EL, respectively. The objectives of this study were to compare the EN and EL flows determined using the EHC method in the growing rat and pig to those determined using the 15N-dilution and G methods and to compare the EN flow estimated by the EHC method with that determined using the 15N-infusion method in the growing pig.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Milk labeled with 15N was collected from a lactating dairy cow housed at the Research Institute for the Biology of Farm Animals (Dummerstorf, Germany) following a 10-d period, during which 10 g of 15N-labeled (15NH4)2 SO4 (96% atom enriched) was infused twice daily into the cow’s rumen via a permanent rumen fistula. The milk was defatted and 15N-labeled casein was isolated from the milk by precipitation with rennin. The 15N-labeled casein was then washed and lyophilized. The N in the casein was shown to be enriched with 0.1125% atom excess, as described in the chemical analyses. A portion of the 15N-labeled casein was guanidinated by reaction with O-methylisourea, as described by Schmitz et al. (1991). This guanidinated portion was mixed with a nonguanidinated 15N-labeled casein at a ratio of 1:1 (wt/wt). The Lys in the resulting mixture was shown to be 40 to 45% guanidinated based on determinations of homoarginine. This mixture (guanidinated 15N-labeled and nonguanidinated 15N-labeled casein) was enzymatically hydrolyzed (pancreatin with an enzyme:substrate ratio of 0.0089, at a pH of 8.0 for 3 h) so that all material would have a molecular weight of less than than 5,000 Da. The enzyme was then inactivated at 90°C for 25 min, and the product freeze-dried. The molecular weight distribution of the peptides in the 15N-labeled guanidinated hydrolyzed casein (15NGH-casein) was determined using an HPLC gel filtration column (Waters Millipore 625 HPLC system and PSK2000SW 60cm column; Milford, MA). The eluting solvent contained 0.1% trifluoroacetic acid and 36% acetylnitrile. The sample was detected using a wavelength of 205 nm. In the 15NGH-casein, 72% of the peptides were found to be between 250 and 5,000 Da, 15% were between 185 and 250 Da, and the remaining material (13%) had molecular weights less than 185 Da.
A semisynthetic diet was prepared with 15NGH-casein as the sole source of protein (15NGH-casein diet). Two further semisynthetic diets were also prepared: an intact casein-based diet (basal diet) and an enzyme-hydrolyzed casein-based diet (EHC diet). The EHC used in the EHC diet was chosen to have a similar molecular weight and AA profile as the 15NGH-casein. Chromic oxide was included in the EHC and 15NGH-casein diets as an indigestible marker. The ingredient compositions of the diets are given in Table 1 and the determined total N and AA compositions of the diets are given in Table 2.
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Rat Study. All protocols for experiments described in this study were approved by the Massey University Animal Ethics Committee. Thirty Sprague Dawley entire male rats with a BW of 179.0 ± 3.0 g (mean ± SEM) were obtained from the Small Animal Production Unit, Massey University, Palmerston North, New Zealand. The animals were housed individually in wire-bottomed cages at 22 ± 2°C with a 12-h light/dark cycle. Fresh water was available at all times. Throughout the trial, the rats were given free access to food for 10 min every hour over 8 h each day, starting at 0800; the final meal of the day was at 1500. The feeders were weighed before and after each meal.
The study was conducted over 15 d, with a 7-d adjustment period followed by an 8-d experimental period. During d 1 to 7, the rats were given access to the basal diet and trained to eat in eight 10-min periods per day as described above. The rats then received the EHC diet during d 8 to 14 in the same manner. On the following day (d 15), the rats were fed the 15NGH-casein diet using the same procedure (i.e., free access to the diet for 10 min every hour). Starting 5 h after the first meal of the day, the rats were killed by asphyxiation with CO2 gas and then decapitated. The body cavity was opened and the last 20 cm of the terminal ileum immediately dissected free, rinsed with distilled water to remove any traces of blood and hair and gently dried with absorbent paper. Care was taken not to apply pressure to the intestine. The digesta were slowly flushed out with deionized water from a syringe. The digesta samples were immediately frozen and stored at -20°C until chemical analysis. The maximum time from euthanasia until the digesta were collected was 4 min. The stomach contents of each rat were checked to ensure that coprophagy had not occurred.
Pig Study. Six Landrace x Large White entire male pigs with a BW of 19.2 ± 0.5 kg (mean ± SEM) were sourced from the Pig Research Unit, Massey University, Palmerston North, New Zealand. The pigs were housed individually in smooth-walled, steel metabolism crates maintained at 22 ± 1°C with free access to water at all times.
Throughout the 15-d trial, the pigs were fed the diets mixed with water (1:1) at 0.10 kg of diet/kg of metabolic BW (BW kg0.75) daily. During d 1 to 7, the pigs were fed the basal diet with half of the daily allowance given at 0800 and the other half at 1700. At the end of this period, the pigs were weighed and the food intake adjusted accordingly for the remainder of the trial. For d 8 to 14, the pigs were fed the EHC diet, and for d 8 and d 9, the daily allowance was fed in two equal portions, at 0800 and 1700. During d 10 to 14, the daily allowance was divided into eight equal portions with one portion fed to the pigs every hour commencing at 0800 daily during d 10 to 11. The feeding times for each pig were staggered by 30 min during d 12 to 14.
On d 15, the pigs were fed the 15NGH-casein diet in the same manner (i.e., staggered hourly meals from 0800). Nine hours after the start of its first meal of the day, each pig was given an i.m. injection of azaperone (Stresnil, Janssen Pharmaceutica, Beerse, Belgium), anesthetized by inhalation of halothane (Fluothane, ICI Pharmaceuticals, Lower Hutt, New Zealand), and killed by intracardial injection of sodium pentobarbitone (Pentobarb 500, Chemstock Animal Health Ltd., Christchurch, New Zealand). The final 20 cm of the terminal ileum were immediately dissected from the body and digesta were collected as described above for the rat study. The digesta were immediately frozen and stored at -20°C until chemical analysis. The maximal time from anaesthetizing a pig until the digesta were collected was 6 min.
Chemical Analysis. The digesta from each rat were freeze-dried. The digesta from groups of five rats were pooled so that a sufficient amount of digesta was available for all chemical analyses, giving a total of six pooled samples from 30 rats. Each digesta pool was carefully mixed and divided into three portions. The digesta sample from each pig was freeze-dried, mixed and then divided into three portions.
One portion of digesta from each sample from the rats and pigs was analyzed for 15 N, 14N, and DM, for calculations using the 15N-dilution method. The second portion from each sample was analyzed for DM, Cr, AA Lys, and homoarginine to allow calculations using the G method. The third portion of digesta from the rat and pig samples was resuspended in distilled water and acidified to pH 3.5 with 6 M sulfuric acid. The samples were stored overnight at 4°C, and then centrifuged at 7,000 x g for 10 min. The supernatants were then ultrafiltered separately using Centriprep-10 ultrafiltering devices (Amicon Inc., Beverly, MA) according to the manufacturer’s instructions. The precipitate from the centrifugation step was added to the retentate (>10,000 Da) from the ultrafiltration step, and the material was freeze-dried. This portion was analyzed for AA, N, Cr, and DM for calculations using the EHC method.
The test diet was analyzed for Cr, DM, total N, 15N, and AA as described below. The chromium contents of the diet and ileal digesta were determined using an Instrumentation Laboratory atomic absorption spectrophometer according to the method described by Costigan and Ellis (1987). Total N for the EHC method was determined in duplicate. The samples were combusted at 1,050°C in oxygen gas. The N was then reduced to N2 by a catalyst and this was measured by a thermal conductivity cell using a Leco FP2000 (Leco Corp., St. Joseph, MI).
The AA composition of the samples was determined as follows: Duplicate samples (5 to 7 mg) were hydrolyzed in 1 mL of 6 mol/L glass-distilled HCl containing 0.1% phenol in glass tubes sealed under vacuum, for 24 h at 110 ± 2°C. Amino acid concentrations were then measured using a Waters ion-exchange HPLC system calibrated against a reference AA mixture. The peaks of the chromatograms were integrated using the dedicated software Maxima 820 (Waters Millipore), which identifies the AA by retention time against a reference AA mixture. Norleucine and lysozyme were used as internal and external standards, respectively, and the weight of each AA was calculated using free AA molecular weights. No corrections were made for losses of AA during hydrolysis.
Cysteine and methionine are partially destroyed during this hydrolysis procedure, so were determined following oxidation of duplicate samples (3 to 4 mg) with 1 mL of performic acid (1 part 30% H2O2 to 9 parts 88% formic acid) for 16 h at 0°C. The samples were then neutralized with 0.15 mL of 50% (wt/wt) HBr before acid hydrolysis. Tryptophan, which is also destroyed during acid hydrolysis, was not determined.
To determine the 15N-enrichment of the diet and digesta for the 15N-infusion method, ground homogenized samples were analyzed using an on-line combined CHN elemental analyzer (EA 1108, Carlo Erba, Italy) and isotope ratio mass spectrometer (IRMS Delta S, Finnigan, Bremen, Germany) for 14N and 15N. This system is accurate to 0.00005% atom. The measured 15N-enrichment of the diet and digesta were corrected for the natural 15 N-enrichment of the preliminary diet and digesta from animals that had not received a 15 N-labeled diet.
Calculations and Statistical Analysis. For the EHC method, the following equation was used:
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where D = digesta, F = feed, and Cr = chromium.
For the G method, the following equations were used:
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where D = digesta, F = feed, and Cr = chromium.
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where D = digesta, F = feed, and Cr = chromium.
For the 15N-dilution method, the following equations were used:
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where total N flow was calculated as described above for Lys:
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Enrichment was in percentage of atom excess.
The data were first tested for homogeneity of variance using Bartlett’s Test (Snedecor and Cochran, 1980), and all variances were found to be homogeneous (P > 0.05). Paired t-test comparisons were carried out using the statistical program SAS (SAS Inst., Inc., Cary, NC) to test for differences in the EL flows determined using the EHC and G methods, and to test for differences in the EN flows determined using the EHC and 15N-dilution methods. An estimate of the total N and AA composition of ileal endogenous excretions reported by Boisen and Moughan (1996b) was used to calculate the EN flow, corresponding to the determined EL flow based on the G method determined in the pig. For the rat, ileal endogenous flows of N and Lys reported in the literature (Butts et al., 1992; Donkoh et al., 1995; Yu et al., 1995) were averaged (average N:Lys ratio was 5.51 µg of N/µg of Lys at a set DMI), and the mean value was used to calculate the ileal EN flow corresponding to the EL flow determined with the G method applied in the rat. The 15N-dilution and EHC methods for determining EN flow were compared using a paired t-test.
Experiment 2
The experiments described in Exp. 2 were conducted under the guidelines for animal research of the German Ministry of Agriculture and were approved by the State Animal Care Committee of the Ministry of Agriculture of Mecklenburg-Vorpommern (Ref. SV-RO ’97).
Six Landrace x Large White castrated male pigs with a BW of 27 ± 0.4 kg (mean ± SEM) were sourced from the Pig Research Unit of the Research Institute for the Biology of Farm Animals, Dummerstorf, Germany. The pigs were housed individually in smooth-walled steel metabolic cages maintained at 22 ± 1°C with free access to water at all times. Throughout the trial, the pigs were fed the diets mixed with water (1:1) at 0.10 kg of diet/kg of metabolic BW (BW kg0.75) daily.
After adaptation to the experimental conditions, the pigs were surgically fitted with catheters into the carotid artery and jugular vein for blood sample collection and 15N-leucine infusion, respectively. The surgery was performed under general anesthesia using ketamine hydrochloride (Ursotamin, Serumwerk, Bernburg, Germany) in combination with diazepam (Faustan, Arzneimittelwerk Dresden Ltd., Dresden, Germany) and a preceding local infiltration with lidocaine hydrochloride (Xylocitin, Jenapharm, Jena, Germany).
From the day of surgery (d 0 of the experiment), the animals received the EHC diet. A blood sample was taken from the carotid catheter of each pig on d 3 (1800) and again at the beginning of d 4 to determine the natural 15N-enrichment in the trichloroacetic acid (TCA)-soluble fraction of the blood plasma of each animal (blank samples).
A sterile, pyrogen-free solution of physiological saline was prepared containing 15 N-Lys-leucine (>95% atom enriched; Chemotrade, Leipzig, Germany) for infusion (syringe pump, Harvard 22, Harvard Apparatus, Holliston, MA) into the jugular vein of the pigs. The 15N-Lys-leucine in the infusion solution was adjusted so that each animal received 10 mg of 15N-Lys-leucine/kg of BW each day at a rate of 2 mL/h starting on d 4 and ending on d 15.
Two 10-mL blood samples were taken from the carotid artery daily (0800 and 1800) throughout the infusion period. The blood was collected into ice-cooled, heparinized tubes and the tubes were immediately centrifuged at 2,000 x g for 15 min. The plasma was then removed, treated twice with an equal volume of 10% TCA solution and centrifuged at 5,500 x g. The supernatant was analyzed for 15N to determine the course of 15N-enrichment in the TCA-soluble fraction of the blood plasma.
Starting with d 11, the daily feed allowance was divided into eight equal portions with one portion fed to the pigs every hour commencing at 0800 daily. The feeding times for each pig were staggered by 30 min from d 13. On d 15, the animals were fed the test diet in the same manner (i.e. staggered hourly meals). Exactly 9 h after the start of its first meal of the day, a final blood sample was taken from each pig and the pig was then euthanased with an injection of sodium ethylbutylthiobarbitural (Brevinarcon, Arzneimittelwerk Dresden Ltd.) given via the jugular vein catheter. The final 20 cm of the terminal ileum was immediately dissected from the body and digesta were collected as described for the pig study in Exp. 1. The digesta were immediately frozen and stored at -80°C until chemical analysis.
The digesta were analyzed for chromium as described above. Freeze-dried duplicate samples of digesta (0.5 g) and TCA-soluble plasma were analyzed for Kjeldahl N. The TCA-soluble plasma Kjeldahl N distillate was placed in small tin cups, dried, and analyzed by elemental analyzer (Carlo Erba) coupled to an isotope-ratio mass spectrometer (Finnigan delta S) to determine the 15N-enrichment.
Digesta were also ultrafiltered as described for Exp. 1 of the study. The precipitate plus retentate fraction was analyzed for total N using the Kjeldahl method, and for AA as described in Exp. 1. The ileal EN and AA flows for the EHC method were calculated as described in Exp. 1.
The data on 15N-enrichment of the TCA-soluble blood plasma fraction were used to calculate the steady state as described by Souffrant et al. (1993).
The proportion of EN in ileal digesta was calculated as:
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where 15Ndigesta and 15Ndeproteinized plasma were the 15N-enrichments measured following 15N infusion in the ileal digesta and TCA soluble plasma, respectively, and Blankdigesta and Blank deproteinized plasma were the 15N-enrichments measured before 15N infusion in the ileal digesta and TCA-soluble plasma, respectively.
Chromium concentrations in digesta and feed were used to calculate total N flows as described above, and EN flow as calculated as the product of total N flow and proportion of EN in ileal digesta.
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Results |
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The rats and pigs appeared healthy throughout the study. The pigs ate all of their allocated dietary allowance throughout the experiment and the rats had a daily feed intake of 14.3 ± 0.2 g (mean ± SEM). During the 8-d period when they were given access to the EHC and 15NGH-casein diets, the growth rates were 4.1 ± 0.2 g/d and 344 ± 31 g/d (mean ± SEM) for the rats and pigs, respectively.
The EL flows determined using the G method in both the rat and pig were lower than those determined using the EHC method (means of 298 vs. 382 µg/g of DMI for the rat, P < 0.05, and 214 vs. 287 µg/g of DMI for the pig, P < 0.01; Table 3). Endogenous N flows determined using the 15N-dilution method in the rat and pig were lower than those determined using the EHC method (means of 1,034 vs. 1,942 µg/g of DMI for the rat, P < 0.001, and 1,011 vs. 1,543 µg/g of DMI for the pig, P < 0.05, Table 3). The ileal endogenous amino acid flows determined using the EHC method in the rat and pig are presented in Table 4.
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). In the pig, the EN flow for the G method did not differ from those determined using either the EHC or 15N-dilution methods (P > 0.05). In the rat, the ileal EN flow for the G method was lower (P < 0.05) than that determined using the EHC method and higher (P < 0.01) than that determined using the 15N-dilution method. Experiment 2
All of the pigs appeared healthy throughout the study. The animals ate all of their allocated dietary allowance throughout the experiment. During the 8-d period when they were given access to the EHC and 15NGH-casein diets, the growth rates were 424 ± 32 g/d (mean ± SEM).
A steady state in 15N labeling was achieved by 6 d after the start of the infusion (d 10 of the experiment; Figure 1) and measurements were started during the 11th d of infusion (d 15). Ileal EN flow determined using the 15N-infusion method was 60% higher (P < 0.01) than that determined using the EHC method (Table 5).
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Discussion |
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The EHC method was used as a baseline for comparison in the present study. When the EHC method is applied more generally to determine ileal endogenous protein flows, the assumption is made that the molecular weight profile of the dietary peptides (i.e., peptide size) does not affect the net endogenous protein flow, but this has not been fully tested. In the present study, however, hydrolyzed casein was fed to all of the animals for all of the experimental treatments, thus any effect of peptide size on ileal endogenous protein flow would be inherent to the one diet and would not affect the comparisons between the methods.
The EHC method has the shortcoming that there is a net underestimation of endogenous AA flow due to a loss of low molecular weight endogenous peptides and AA in the ultrafiltrate. Studies have attempted to quantify the extent of this loss by ultrafiltering digesta collected from animals given a protein-free diet and quantifying the AA present in the ultrafiltrate. In a controlled study, Moughan and Schuttert (1991) found that approximately 10% of the amino N from total digesta was present in the ultrafiltrate. It should be noted, however, that feeding protein or an enzyme-hydrolyzed protein (such as casein) to an animal may affect the composition as well as the amount of endogenous excretion, and that higher or lower than normal amounts of free AA and small peptides may occur during protein-free feeding. It is difficult to accurately determine the amounts of small (<10,000 Da) amino molecules in the ileal digesta of animals receiving an EHC-based diet, although the concentration of such molecules at the terminal ileum is expected to be low. It is concluded that the EHC method applied here may lead to some (unquantified) underestimation of total endogenous AA flow at the end of the ileum and the results need to be interpreted accordingly.
In Exp. 1 of the present work, the mean EL flows were 22 to 25% lower when determined using the G method in comparison with those determined using the EHC method. This was true for both the rat and pig. This suggests that the G method considerably underestimates EL flow. The reason for this is not clear. Most of the assumptions that are made when the G method is used to determine ileal EL flow have been tested and appear to be valid as reviewed by Hodgkinson and Moughan (2000). The 15NGH-casein used in the present study was partially guanidinated as opposed to fully guanidinated. A comparison of ileal EL flows determined using partially guanidinated protein with those determined using fully guanidinated protein demonstrated no differences in the results obtained using the two methods (Moughan and Rutherfurd, 1990). Thus, partial guanidination is not expected to explain the lower Lys flows determined in the present study with the G method than those determined using the EHC method.
A further assumption that is made when a partially guanidinated protein is used with the G method is that the transformation of Lys to homoarginine in the guanidinated protein occurs randomly, so that the protein is homogeneously labeled with homoarginine. Siriwan et al. (1994) conducted sequential in vitro proteolysis of guanidinated casein and soybean protein followed by AA analysis of the digested fractions. The guanidinated proteins were shown to contain constant ratios of homoarginine to other AA in all of the fractions, thus supporting this assumption.
One area that has not been fully investigated is the possible effect of homoarginine on protein digestion and metabolism. Drescher et al. (1994) found a lower true N digestibility for guanidinated diets than for nonguanidinated diets. However, in the present study, any effect of the homoarginine in the diet on protein digestion and metabolism and which could affect EL flow determined using the G method, would also have been reflected in the results from the EHC method. A comparison of the EL flows determined using the EHC and G methods has not been made previously.
In the rat and pig, ileal EN flow determined using the 15N-dilution method (Exp. 1) was significantly lower than that determined using the EHC method. Given that the EHC method is expected to underestimate ileal EN flow, these results suggest that, as for the G method, the 15N-dilution method underestimates ileal EN flow. The use of 15N-labeled dietary protein to estimate EN flows assumes the absence of discrimination between the absorption of labeled and unlabeled AA. It is also assumed that the endogenous protein that is secreted into the gut does not become labeled during the course of the trial. However, it is known that the enterocytes take up a proportion of the labeled dietary N and resecrete it rapidly into the intestine, which will result in some endogenous protein being accounted for as unabsorbed exogenous protein. Following the ingestion of a single 15N-labeled meal in the growing pig, Leterme et al. (1996) found the presence of 15N in pancreatic enzymes within 50 min, in the bile within 90 min, and in the intestinal mucus within 4 h of the meal. In the present study, some of the labeled dietary AA may have been absorbed and resecreted in the period of time between consumption of the labeled dietary AA and sampling of the digesta. Resecreted AA are endogenous, but would have been labeled with 15N and thus during the analysis would have contributed to the estimate of undigested dietary AA. This may explain the underestimation of ileal EN flow observed in the present study with the 15N-dilution method. Digesta were collected 5 and 9 h after the first meal of the 15N-labeled test diet for the rats and pigs, respectively, in an attempt to minimize the extent of recycling; however, recycling could still have been significant.
In Exp. 2 of the study, the ileal EN flows determined using the 15N-infusion method were significantly (P < 0.01) greater than those determined using the EHC method. This is in contrast with the results reported by Schulze et al. (1995), who also compared the ileal EN flows determined using the EHC and 15N-infusion methods and found no significant difference between EN flows determined using the two methods.
It should be noted that in Exp. 2 of this study, 15N-leucine was infused into the animals. It was assumed that other AA would have been labeled by transamination. However, the 15N label is not transferred to Lys and threonine, and the isotopic enrichment of other AA has been shown to be less than that of the labeled leucine (de Lange et al., 1992; Lien 1997a, b). The use of mixtures of 15N-labeled AA has been suggested (Reeds et al., 1997) and may avoid this problem.
Another concern with the use of the 15N-infusion method is the choice of an appropriate precursor pool (Moughan et al., 1992). The TCA-soluble blood plasma is the most commonly used pool to indicate 15N-enrichment of the gut endogenous material with the assumption made that the free AA in the TCA-soluble pool are used for the synthesis of body protein, and thus for most of the endogenous secretions into the digestive tract. For small intestinal mucosa secretions, at least absorbed dietary AA are used directly for protein synthesis (Alpers, 1972). The 15N-enrichment of the TCA-soluble blood plasma fraction is lower than that in pancreatic secretions and in urinary urea plus ammonia (Souffrant et al., 1993). Thus, the use of plasma enrichment may underestimate the enrichment of EN secreted into the intestinal lumen leading to an overestimation of endogenous protein excretion (Lien et al., 1997b). Therefore, in Exp. 2 of the present study, there may have been an overestimation of the ileal EN flows determined using the 15N-infusion method.
Given that in Exp. 1 of this study, the EN flows determined using the 15N-dilution method were significantly lower than those determined using the EHC method, whereas in Exp. 2 of this study, the flows, determined using the 15N-infusion method, were significantly higher than those determined using the EHC method, it would appear that ileal EN flows determined using the 15N-dilution method are likely to be lower than those determined using the 15N-infusion method. Leterme et al. (1994) compared EN flows determined for the pig using the 15N-infusion method with those determined using the 15N-dilution method and reported that the EN flow determined using the 15N-infusion method was higher than that using the 15N-dilution method.
Ileal endogenous total N flow and flows for AA other than Lys can be calculated using the G method. This is generally done by assuming that ileal endogenous excretions have a constant N and AA composition. In a review, Boisen and Moughan (1996b) reported a high degree of consistency in N and AA composition of ileal endogenous secretions. Souffrant et al. (1997) reported ileal EN flows in the pig determined using both the 15N-perfusion method and the G method. The EN flows calculated with the G method were quantitatively greater than those determined with the 15N-perfusion method, although no statistical comparison was reported. Roos et al. (1994) determined the true digestibility of casein doubly labeled with homoarginine and 15N. The digestibility of casein was determined at 3, 6, and 12 h following the meal. The digestibilities determined with the G method were significantly higher than those determined using 15N at all sampling times. This corresponds to greater endogenous flows determined with the G method than the 15N-dilution method. It was suggested that the difference in digestibility between the two methods may have been due to recycling of the 15N label, resulting in an underestimation of the endogenous component with the 15N-dilution method (Roos et al., 1994).
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Footnotes |
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2 Correspondence and current address: Instituto de Producción Animal, Universidad Austral de Chile, Casilla 567, Valdivia, Chile (phone: 056-63-221660; fax: 056-63-221460; E-mail: shodgkin{at}uach.cl).
Received for publication April 4, 2003. Accepted for publication June 6, 2003.
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