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Nutrient Metabolism

Route of Tracer Administration Does Not Affect Ileal Endogenous Nitrogen Recovery Measured with the ¹⁵N-Isotope Dilution Technique in Pigs Fed Rapidly Digestible Diets^1,2

C. A. (Carina) Steendam³, Martin W. A. Verstegen, Seerp Tamminga, Huug Boer, Marianne van ’t End, Berthe Verstappen^*, William R. Caine and G. Henk Visser^*

Animal Nutrition Group, Wageningen Institute of Animal Sciences (WIAS), Wageningen University and Research Centre, 6700 AH Wageningen, The Netherlands; Olds College Centre for Innovation, Olds, AB, Canada, T4H 1R6; and ^* Centre for Isotope Research (CIO), University Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands

³To whom correspondence should be addressed. E-mail: Carina.Steendam{at}wur.nl.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The ¹⁵N-isotope dilution technique (¹⁵N-IDT), with either pulse-dose oral administration or continuous i.v. administration of [¹⁵N]-L-leucine (carotid artery), both at 5 mg/(kg body weight · d), was used to measure ileal (postvalve T-cecum cannula) endogenous nitrogen recovery (ENR) in pigs (9 ± 0.6 kg). Diets were cornstarch, enzyme-hydrolyzed casein with no (control) or high (4%) content of quebracho extract (Schinopsis spp.) rich in condensed tannins. Blood was sampled from a catheter in the external jugular vein. Mean plasma ¹⁵N-enrichment at d 8–10 was higher (P = 0.0009) after i.v. than after oral administration [0.0356 vs. 0.0379 atom% excess (APE)]. Plasma ¹⁵N-enrichment for i.v. infused pigs was 0.01117 APE higher (P < 0.0001) and for orally dosed pigs 0.0081 APE lower (P < 0.0001) at 11 h postprandial compared with 1 h postprandial. Apparent ileal N digestibility was higher (P < 0.0001) for the control (85.5%) than for the quebracho diet (69.5%). ENR was calculated from the ratio of ¹⁵N-enrichment of plasma and digesta. The ENR for the quebracho diet was 300% higher than for the control diet (6.03 vs. 1.94 g/kg dry matter intake, P < 0.001). The real N digestibility (92.2 ± 0.4%) was equal for both diets (P = 0.1030) and both tracer methods (P = 0.9730). We concluded that oral administration of [¹⁵N]leucine provides reasonable estimates of ENR in pigs fed semipurified diets with high or low content of tannins; however, one must be careful in extrapolating this conclusion to studies with other protein sources or feeding frequencies.

KEY WORDS: • ¹⁵N-isotope dilution technique • oral label administration • first-pass uptake • endogenous N • quebracho tannin

In human (1) and animal nutrition (2), the use of real rather than apparent ileal nitrogen digestibility (AD-N)⁴ is preferred. Values for real ileal N digestibilities (RD-N) are higher than those for AD-N because they are corrected for endogenous nitrogen recovery (ENR), which can vary among diets (2–4). The present study was done in pigs but ENRs were also reported in other species (5,6). One of the methods used to estimate ENR at the terminal ileum in pigs is the ¹⁵N-isotope dilution technique (¹⁵N-IDT) (7,8). Pigs are labeled with a ¹⁵N-isotope and ENR is calculated from the ratio of ¹⁵N-enrichment of trichloroacetic acid (TCA)-soluble plasma and of ileal digesta.

The original method involved the continuous i.v. infusion of a ¹⁵N-labeled amino acid, usually [¹⁵N]-L-leucine (7–10), to steady-state ¹⁵N-enrichment of plasma and body pools. A synthetic amino acid (AA) is completely absorbed before the end of the ileum (11); therefore we hypothesize that the crystalline [¹⁵N]-L-leucine tracer is also completely absorbed and can be administered with the feed. This is less invasive and would include labeling of ENR synthesized directly from first-pass uptake of AA in the splanchnic region. First-pass uptake of dietary N or AA by the splanchnic region was shown to range from 15 to 18% in the fed state (12,13). Stoll et al. (12) estimated that 10% of dietary N was resecreted into the gut lumen after first-pass uptake. With the exception of a preliminary report (14), ENR in pigs, estimated with oral administration of [¹⁵N]leucine, has not been reported.

Another concern with the ¹⁵N-IDT is the necessity to reach steady state (2). Discontinuous oral [¹⁵N]-leucine administration could possibly influence steady state. On the other hand, the flux of unlabeled N from feed will also cause fluctuations in plasma ¹⁵N-enrichment when the tracer is continuously infused (15). As such, time of blood sampling is critical to both approaches.

ENR is influenced by many factors including dietary factors. One of these is the presence of dietary condensed tannins such as those found in quebracho extract (from the heartwood of Schinopsis spp.). The objective of the present study was to compare ENR in pigs calculated from ¹⁵N-enrichment of digesta and ¹⁵N-enrichment of TCA-soluble plasma after either pulse-dose oral administration or continuous i.v. administration of [¹⁵N]-leucine for diets causing low (control casein diet) or increased ENR [control + quebracho-extract rich in condensed tannins (Schinopsis spp.)].

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Experimental design. Treatments were allocated according to a repeated-measures design (16). Pigs (n = 12) were divided into 4 groups of similar mean body weight (BW). Treatments consisted of method of tracer administration [i.e., continuous intravenous (i.v.) vs. pulse-dose oral administration (oral)] of the synthetic [¹⁵N]-L-leucine label, and diet (control vs. control + 4% quebracho extract). Method of tracer administration (i.v. or oral) was allocated to each pig. Diet was allocated to repeated measures within pigs. Each experimental period lasted 10 d. The method of tracer administration was kept constant and diets were switched after the first experimental period according to a crossover design.

    Pigs and pig care. Ethical approval for this study was given by the TNO-committee for Animal Welfare. At the conclusion of the study, pigs were inspected by the veterinary surgeon of the Ethics Committee and assigned to a second experiment. Crossbred [(Dutch Landrace x Finnish Landrace) x Great Yorkshire] barrows (n = 12) were obtained from a commercial breeding farm (W v.d. Brink, Putten). On arrival, pigs (weight 9 ± 0.6 kg) were housed individually in transparent smooth-walled metabolism cages (0.8 x 0.9 m) with a plastisol surface (Tenderfoot). The cages were located in a temperature-controlled room (22–24°C, mean relative humidity 40%), with illumination from 0700 to 2100 h. The health of the pigs was checked daily and in case of abnormalities, a veterinary surgeon was consulted. Removal of pigs and the reasons for removal are explained in the Results.

    Adaptation and surgery. The general outline of the experiment is summarized in Table 1. After arrival, the pigs were acclimated to the experimental conditions. During the first 6 d, the pigs were fed the commercial diet they had received at the breeding farm. On d 6–10, pigs were fitted with a postvalve T-cecum cannula (17). After pigs recovered from surgery, diets were gradually changed to a casein-based diet (Table 2). During the adaptation period, pigs were fed at 0800 and 1600 h a mixture of feed and water (1:1.25, wt:wt) at 2.6 times energy for maintenance (18) as previously described by Schulze et al. (9).

    Experimental diets. On d 0 of the experimental periods, pigs were fed the experimental diets (Table 2) at 0800 and 2000 h in a mixture of feed and water (1:1.25, wt:wt) at 2.6 times energy for maintenance (18). The control diet contained enzyme-hydrolyzed casein (EHC, Quest International, Hyprol 8360, MW < 5 kDa) as the sole N source and Cr₂O₃ (1 g/kg) was added as an indigestibility marker (Table 3). The quebracho diet was formed by adding quebracho extract (Unitan Saica, Quebracho Superior ATO, 120 g/kg catechin equivalents, 650 g/kg total phenols, 9 g/kg crude protein) to the control diet at 4% of the feed intake. Total intake from the quebracho diet was 104% of the feed intake from the control diet.

    Sampling. Blood samples of 10 mL (Sarstedt monovette 10-mL heparinized tubes) were collected at 0930 h on d 0 and at 1300 h on d 2, 5, and 7, of period 1 and d 3, 5, and 7 of period 2. On d 8–10 of each period, blood samples were collected at 0900, 1400, and 1900 h, corresponding to 1, 6, and 11 h after the morning meal.

Digesta were collected from the morning feeding (0800 h) to just before the evening feeding (2000 h) on d 8–10 of both periods. Collection bags (Stomacher) were attached to the cannulas and changed at least every hour; digesta were immediately stored at –20°C.

    Sample preparation and analyses. After collection, blood samples were centrifuged immediately (10 min, 1000 x g, 4°C). Plasma was stored at –20°C for determination of ¹⁵N-enrichment of TCA-soluble plasma. Before analysis, plasma samples were deproteinized by adding 200 µL TCA (400 g/L; 2.463 mol/L) to 1 mL of plasma. TCA-soluble plasma was neutralized with NaOH, freeze-dried, and sent to the Centre for Isotope Research (CIO) for duplicate determination of ¹⁵N-enrichment. Samples were analyzed using a Carlo Erba NA1500 combustion system, as well as a Europa Scientific 20–20 Isotope Ratio Mass Spectrometer. In each batch, International Atomic Energy Agency standards that covered the observed enrichment range between background and high enrichment (i.e., N1, N2, 310A and 310B standards) were applied. For all subsequent calculations, values for ¹⁵N/¹⁴N isotope ratios for each sample were converted to ¹⁵N concentrations.

Digesta were pooled within pig and period and then sampled for dry matter (DM) determination. The remainder was freeze-dried, ground with a mortar and analyzed in duplicate for DM, ash, Kjeldahl-N, Cr₂O₃ and ¹⁵N-enrichment. The DM was determined according to the International Organization for Standardization (ISO) 6496 (21) and Kjeldahl-N according to ISO 5983 (22). The Cr₂O₃ was analyzed as described by Jansman et al. (23). Total phenolics of the quebracho extract were determined with a Folin-Denis assay according to AOAC official method of analysis 952.03 (24) after boiling the sample in a mixture with water under reflux for 2 h. Catechin equivalents were determined with vanillin-sulfuric acid according to a method adapted from Kuhla and Ebmeier (25). Samples were boiled under reflux for 15 min instead of 10 and subsequently centrifuged for 10 min at 900 x g, instead of filtered.

For ¹⁵N determination, 4 mg of freeze-dried digesta, containing 100 µg of N, was weighed into tin cups. The samples were then treated similarly to the plasma samples.

    Calculations and statistics. All calculations and statistical analyses were performed using SAS v6.12 (26). Statistical analyses and linear curve fitting were done using the General Linear Model procedure of SAS. Least-square means were tested with the Tukey’s t test. Nonlinear curve fitting was done with the MODEL procedure [Marquardt method, (26)]. Values were expressed as least-square means ± SE. Differences were considered significant at P < 0.05.

    Plasma ¹⁵N-enrichment. Plasma ¹⁵N-enrichments (E_pl) of each sample were converted from atom% to atom% excess (APE); i.e., the individual ¹⁵N-enrichment values (atom%) minus the overall baseline ¹⁵N-enrichment (E_pl-base, mean of all pigs in atom%) before the administration of [¹⁵N]-L-leucine. For days with multiple samples, mean plasma ¹⁵N-enrichment per day (E_pl,d) was calculated as the weighted mean of the first time span (h 1–6) and second time span (h 6–11) after the morning meal:

(1)

Enrichment at h 6 is included twice to give a better fit of the data. It is assumed that the effects of h 0–1 and h 11–12 can be ignored.

The overall mean plasma enrichment for d 8–10 (corresponding to the days on which digesta collection took place, E_pl,d8–10) was calculated per pig as the weighted mean of E_pl(d 8), E_pl(d 9), and E_pl(d 10).

The effect of diet on plasma ¹⁵N-enrichment was tested with model 2:

(2)

where µ is the mean, T_i is the effect of method of tracer administration (i = 1,2), and S_j is sequence of dietary treatment (j = 1,2). T_i, S_j, and T_i x S_j are tested against Pig_k(T_i x S_j) as an error term (k = 1–12), D_l is the effect of diet (l = 1,2), P_m is the effect of period (1,2), and e_ijklmn is the overall error (m = 1–24). Pig, sequence, and period effect were added as correction factors.

When the effect of diet was not significant, variables corresponding to plasma ¹⁵N-enrichment on d 8–10 of both periods were tested with model 3:

(3)

where µ is the mean, T_i is the effect of method of tracer administration (i = 1,2) using the variation between pigs within a tracer group, Pig_j(T_i), as an error term (j = 1–12), P_k is the effect of period (k = 1,2), and e_ijkl is the overall error (l = 1–24). Pig effect, period effect and their interactions were added as correction factors.

The effect of hour or day of plasma sampling on plasma ¹⁵N enrichment or ENR, was tested with a paired t test (e.g., ENR at h 11 – ENR at h 1) within the appropriate model (2 or 3).

The relative importance of effects was tested by paired t tests for differences between absolute values of the following variables: Day effect = E_pl(d 10) – E_pl(d 8) (per pig per period), Meal effect = E_pl(h = 1, mean of d 8–10) – E_pl(h = 1, mean d 8–10) (per pig per period). Tracer effect = E_pl,d8–10(infusion group) – E_pl,d8–10(oral group) (per period).

Gut N-disappearance (GND, fraction) was calculated as:

(4)

where E_pl is the plasma ¹⁵N-enrichment in APE and i is the actual infusion rate or dosage (mg/(kg BW · d).

    Digesta. The ileal dry matter (DM) flow of digesta throughout the day (g/d) was calculated from data on dry matter intake (DMI; g/d), and chromic oxide content of digesta and feed (mg/kg DM):

(5)

Apparent ileal N digestibility (AD-N, %) was calculated from N contents in feed and in ileal digesta:

(6)

with N_digesta in g/g DM and DM_flow and N_intake in g/d.

    Endogenous nitrogen recoveries. The dilution factor (DF) is defined as

(7)

where E_digesta is the ¹⁵N-enrichment per pig in APE of digesta (pooled sample from d 8–10) above enrichment of feed and E_pl,d8–10 is the mean ¹⁵N-plasma enrichment per pig of d 8–10 in APE above baseline. The endogenous N recovery (ENR g/d or g/kg DMI) was calculated from the total N flow and the dilution factor:

(8)

The real N digestibility was calculated by correcting the AD-N for the endogenous N recoveries:

(9)

with N_digesta in g/g DM and DM_flow, ENR and N_intake in g/d. Effects of diet, method of tracer administration, and sequence of dietary treatment in periods 1 and 2 were tested with model 2.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Pigs. After a few meals, the pigs readily ate the experimental diets. One pig was killed on d 9 of period 1 because it had a torsion in the intestine. In period 2, it was replaced by a spare pig of the same age and breed. Another pig was killed on d 9 of period 2 because of problems with the cannula.

Growth depended on the diet and on the age of the pigs. In period 1, the daily growth was 304 ± 16 g/d for pigs fed the control diet and 260 ± 15 g/d for pigs fed the quebracho diet. Corresponding values for period 2 were 283 ± 12 and 249 ± 43 g/d, respectively.

Plasma ¹⁵N-enrichment

    Diet effect. There was no effect of diet nor interactions between method and diet for the plasma ¹⁵N-enrichment, and statistical analysis was continued with model 3.

    Method effect (oral vs. i.v.). Plasma ¹⁵N-enrichment (E_pl,d8–10) in the infusion group was higher (P = 0.0009) than in the oral group (Table 3).

    Steady state (day effect). Only for the i.v. group in period 2 was steady state reached at d 8. Plasma ¹⁵N-enrichments at d-9 did not differ from those of d 10 (E_pl,d10 –E_pl,d9 = 0.00038 ± 0.00032 APE, P = 0.2573), showing that by d 9, plasma steady state was reached for all groups in both periods.

    Time of blood sampling (meal effect). Plasma ¹⁵N-enrichment (d 8–10) varied between hours (1–11 h) after the morning meal (Fig. 1). At 11 h postprandial, ¹⁵N-enrichment was 0.0117 ± 0.0009 APE higher for i.v. infused pigs (P < 0.0001) and 0.0081 ± 0.0009 APE lower for orally dosed pigs (P < 0.0001) compared with 1 h postprandial. The direction of the meal effect differed with tracer method (P < 0.0001). The absolute value of the meal effect was larger for i.v. infused pigs than for orally dosed pigs (P = 0.0174, Table 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 1 15N-enrichment of deproteinized plasma (APE) in pigs at 1, 6, or 11 h after the morning meal on d 8–10 in experimental periods 1 (A) and 2 (B), Values are least-square means ± pooled SE. Depending on day and period n = 4 or 5 for the infusion group and 5 or 6 for the oral group.

Fluctuations in plasma ¹⁵N-enrichment after feeding resulted in differences in calculated ENR when using plasma samples taken at different hours. Six hours after feeding, the ENR was 14% higher (i.v., P = 0.0007) or 20% lower (oral, P = 0.0001) than 1 h after feeding; 11 h after feeding, this was 27% higher (i.v., P = 0.0001) and 26% lower (oral, P = 0.0001) than 1 h after feeding.

Digesta ¹⁵N-enrichments, apparent and real N digestibility, and ENR did not differ between methods of tracer administration. Digesta ¹⁵N-enrichment was higher (P < 0.0001) for the quebracho diet (0.0267 ± 0.0006 APE) than for the control diet (0.0179 ± 0.0006 APE). The dilution factor was also higher (P < 0.0001) for the quebracho diet (0.73 ± 0.009, fraction) than for the control diet (0.49 ± 0.010). Apparent ileal N digestibility of the control diet was higher (P < 0.0001) than that of the quebracho diet (Table 5). The calculated overall ENR (g/d or g/kg DMI) was entirely dependent on the diet. Pigs that were fed the quebracho diet had ENRs that were 300% higher (P < 0.0001) than those of pigs fed the control diet. Finally, RD-N did not differ between the diets (P = 0.1030).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The first concern with the original method is the choice of the precursor pool or reference pool. For quantitative measurements of ENR, the enrichments of the real precursor pools, i.e., AA bound to intracellular tRNA (31), have to be measured as well as the ratio in which the different precursor pools (e.g., intestinal mucosa, pancreatic secretions, saliva) contribute to the total ENR (2). Even if it were possible to fulfill the first prerequisite, which is very difficult and laborious [see Assimon and Stein in (31)], it is impossible to fulfill the second. Different pools contribute in different ratios, depending on diet (2) and protein turnover rate (29). The TCA-soluble blood plasma pool has frequently been used as the reference pool. Levels of ¹⁵N-enrichment were found to be very similar among TCA-soluble plasma, pancreas, small intestine, and liver by some authors (31–33) but not by others (34–36). Other pools, such as the ¹⁵N-enrichment of mucin, were used (29,30), but this does not solve the problem that one pool cannot be used to represent all.

Luminal AA of dietary origin contribute largely to endogenous protein synthesis (29). Alpers (37) showed that epithelial cells in the tip of gut villi had a preference for the use and inclusion of luminal AA. Stoll et al. (12,13) showed incorporation of dietary protein into mucosal and hepatic protein on first-pass utilization. They also estimated that 10% of dietary N might be resecreted into the gut lumen after first-pass uptake. By using the ¹⁵N-IDT with i.v. labeling and TCA-soluble plasma as a reference pool, resecretion of dietary N into the gut lumen is neglected. Thus, even though the ¹⁵N-IDT in itself is believed to overestimate ENR, the incorporation of unlabeled lumen N into endogenous protein will lead to underestimation of ENR. The use of portal blood plasma as the reference pool would partially solve this problem because enrichment is closer to that of splanchnic tissue pools (28). However, it still does not include all ENR directly synthesized from luminal AA. The use of oral label administration will label luminal AA as well as splanchnic tissues and this might be an improvement to the original method.

Another major concern with the ¹⁵N-IDT is the uneven distribution of ¹⁵N from leucine to other amino acids (and urea) via trans- and deamination.

Non-AA-N comprises up to 50% of total N in TCA-soluble blood plasma (27,38). On the other hand, non-AA-N in endogenous recoveries was estimated at a maximum of 25% (39,40). Data on plasma urea enrichment in the literature are not conclusive. High enrichment of urea was found by De Lange et al. (8,27) in urine and blood. Leterme and co-workers (29), on the other hand, found no difference in enrichment of total N in TCA-soluble plasma compared with -AA-N 2 h after feeding; 6 h after feeding, enrichment of total N was 9% lower.

Differences in ¹⁵N-labeling of different amino acids was shown by several authors (27,28,30,41). Estimates of ENR for highly digestible diets seem to be little affected by heterogeneity (27,41). Quantitative estimates of RD-N and ENR for other diets may be less accurate. In the studies mentioned above, the ranking of the diets for ENR was similar regardless of the method (N dilution or AA dilution) that was used.

In summary, the ¹⁵N-IDT can be used for ranking diets with respect to ENR (27–29). The use of oral label administration might be an improvement to this method because ENR synthesized from luminal absorbed N is included in the measurement.

Effect of method of tracer administration

    Plasma ¹⁵N-enrichment. Mean plasma ¹⁵N-enrichment for oral [¹⁵N]-L-leucine administration was 6% lower (P < 0.0001) than for i.v. infusion of the isotope. There was no difference in labeling of digesta (P = 0.6341) between oral administration and infusion. This suggests that all orally administered label was absorbed in the gastrointestinal tract. Huisman et al. (11) also reported a total uptake of dietary [¹⁴C]methionine.

We used a linear approach to calculate daily mean plasma ¹⁵N-enrichment. A (negative) peak value for plasma enrichment can be expected between 2 and 3 h postprandial (42–44). This means that plasma enrichment may be overestimated (i.v.) or underestimated (oral). It is unlikely that plasma ¹⁵N-enrichment after oral administration is higher than after i.v. infusion. Therefore, we conclude that 0–6% of dietary N was retained within the gut or the splanchnic region. This is lower than the 15% of dietary AA incorporated into mucosal and hepatic protein and the 18% of dietary total N first-pass uptake found in the fed state in studies by Stoll et al. (12). In our study, tracer administration was more than 8 d instead of 6 h (12), resulting in high recycling rates. Meals in our study were given only once every 12 h, compared to hourly by Stoll et al. (12). After 12 h of fasting, total (first + second pass) uptake of leucine by the splanchnic region was already decreased (45) to 2–3% of delivery. In rats, first-pass uptake of [¹⁴C]-L-leucine tracer was almost absent after 15 h of food deprivation (46). Therefore, we should be careful when applying oral label administration with different meal patterns.

When feeding diets containing intact protein 2 times/d, Van Leeuwen et al. (14) reported that plasma ¹⁵N-enrichment at 1400 h was 19% lower (P > 0.05) after oral administration (0.0227 ± 0.0012 APE) of [¹⁵N]-leucine than after i.v. administration (0.0281 ± 0.038 APE). Furthermore, labeled [1-¹³C]-leucine present in casein gave higher (P > 0.05) splanchnic uptake (0.35 ± 0.11) than [1-¹³C]-leucine + free amino acids (0.28 ± 0.08) (47). Finally, in the present study, adding quebracho extract to the basal diet slightly increased GND (6.6% vs. 5.1% for the control and quebracho diet, respectively, P = 0.70). This indicates that splanchnic uptake might be somewhat influenced by type of diet but significant effects were not shown.

    Endogenous nitrogen recoveries. Despite a 6% difference in plasma ¹⁵N-enrichment (P < 0.001) and similar digesta ¹⁵N-enrichments (P = 0.63), calculated ENRs were similar for oral and i.v. administration (P = 0.95). There is considerable variation between pigs, both with respect to digesta enrichment as well as N content of the digesta. This can be caused by differences in digesta flow and chromic oxide recovery.

As stated above, Van Leeuwen et al. (14) found no significant differences in plasma ¹⁵N-enrichments. Digesta ¹⁵N-enrichment of the oral group (0.038 ± 0.0031), however, was clearly higher (P < 0.05) than digesta ¹⁵N-enrichment of the i.v. group (0.013 ± 0.0006 APE). Thus, for the oral group, DF ranged from 1.03 to 2.41 with a mean of 1.69. Although blood sampling took place only at 1400 h, these findings confirm that one must be careful in extrapolating our conclusion regarding oral label administration to studies with other protein sources.

    Steady state (day effect) and meal effect (hour effect). The steady state of plasma ¹⁵N-enrichment is often mentioned as a basic requirement for the ¹⁵N-IDT (2). However, the enrichment of digesta follows that of the precursor pool within a short time due to rapid turnover and recycling [(48); Van Bruchem, unpublished data, see (4); Moughan and Buttery, unpublished data, see (49)]. Although the plasma enrichment is not in steady state by d 8, the dilution factor will not vary greatly.

Fluctuations in plasma ¹⁵N-enrichment after feeding resulted in differences in calculated ENR when using plasma samples taken at different hours. This effect is confirmed by the literature (29). When using TCA-soluble total-¹⁵N, the ENR measured 6 h after the meal was 9% lower than 2 h after the meal (i.v.). Therefore, when comparing literature values, differences in the time of blood sampling should be taken into account. Each new experiment should start with a pilot study to establish the pattern of changes in blood plasma enrichment for each diet that is to be tested. Samples taken right after feeding should be frequent (every 15 or 30 min). From the pilot curve, the most appropriate sampling times can be chosen.

    Level of ENR and effect of quebracho. The level of estimated ENR in the control group (1.94 g/kg DMI) was lowcompared with literature values of 2.5–5.9 g/kg DMI (9,44,50–52) for studies with similar levels of dietary EHC. Quebracho extract clearly increased estimated ENR but it did not affect RD-N (Table 5). This is confirmed by studies of fecal RD-N in rats fed quebracho (53) or tannic acid (54).

In conclusion, when feeding highly digestible diets, estimates of ENR were not different between oral administration and i.v. infusion of tracer. However, when using different feeding frequencies or forms of dietary protein, there may be an influence of route of tracer administration. There is 30% variation in estimates of ENR, related to time of blood sampling after a meal. Adding 4% quebracho extract to the diet increased estimated ENR by 300% but did not affect RD-N.

	ACKNOWLEDGMENTS

 
We thank Piet van Leeuwen for performing the surgeries, Dick van Kleef and Kasper Deuring for technical assistance, and Erik-Jan de Jong for assistance with chemical analyses.

	FOOTNOTES

 
¹ Presented in part at the Third International Workshop on Antinutritional Factors in Legume Seeds and Rapeseed, July 8–10, 1998, Wageningen, The Netherlands [Steendam, C. A., de-Jong, E. J., Mattuzzi, S. & Visser, G. H. (1998) Comparison of three methods for the measurement of the endogenous N-flow at the terminal ileum of pigs, as affected by dietary quebracho extract. Recent advances of research in antinutritional factors in legume seeds and rapeseed. EAAP publication no. 93: 335–340 (abs.)].

² Supported by the Netherlands Organization for Scientific Research (NWO).

⁴ Abbreviations used: AA, amino acid; AD-N, apparent nitrogen digestibility; APE, atom% excess; BW, body weight; DF, dilution factor; DM, dry matter; DMI, dry matter intake; EHC, enzyme-hydrolyzed casein; ENR, endogenous nitrogen recovery; GND, gut N-disappearance; ISO, International Organization for Standardization; ¹⁵N-IDT, ¹⁵N-isotope dilution technique; RD-N, real nitrogen digestibility; TCA, trichloroacetic acid.

Manuscript received 27 April 2004. Initial review completed 28 June 2004. Revision accepted 27 August 2004.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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