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Validity of specifically applied rare earth elements and compartmental models for estimating flux of undigested plant tissue residues through the gastrointestinal tract of ruminants

W. C. Ellis^*,1, M. J. Wylie^,2 and J. H. Matis

^* Department of Animal Science, Texas A&M University, College Station 77843-2471; and Department of Animal Science, University of Wisconsin, Madison 53706; and Department of Statistics, Texas A&M University, College Station 77843-2471

¹ Correspondence:
(phone: 979-845-5063; fax: 979-845-5292; E-mail:
w-ellis{at}tamu.edu).

	Abstract

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The validity of using rare earth elements as flow markers of undigested residues was evaluated by comparing mean gastrointestinal residence time (GMRT) of rare earths specifically applied to cottonseed hulls (CSH) to that of the indigestible fiber of CSH. Feces were collected from five lambs fed a mineral supplemented diet of CSH containing 52 g CP/kg DM and five lambs fed a CSH plus cottonseed meal diet (CSH+CSM) containing 123 g CP/kg DM. Rare earth elements (La, Yb, and Tb) specifically bound to CSH were included in the diet for a 5-d period and then deleted from the diet for a 3-d period. Following the last fecal collection, lambs were slaughtered for collection of digesta from segments of the gastrointestinal tract. Potentially indigestible NDF (PIF) was determined in diets and digesta from each segment of the gastrointestinal tract. Mean turnover rate, time delay, and GMRT for each rare earth element was estimated by fitting an age-dependent compartment model to profiles of markers appearing in the feces (compartmental model-marker method, CMM). The GMRT also was computed by the indigestible entity pool dilution method (IEPD) as grams of PIF in sampled segment/mean intake rate of PIF proceeding slaughter, g/h. The GMRT computed by the CMM and the IEPD methods did not significantly (P < 0.05) differ (99.6 vs 94.8 h and 58.9 vs 59.5 h for CMM vs IEPD and CSH and CSH+CSM diets, respectively). Regression of GMRT estimated for rare earths vs PIF yielded a highly significant regression (P = 0.001) with a regression coefficient of 0.94 ± 0.016. It was concluded that rare earth elements applied to specific feeds are valid flow markers for the undigested residues derived from such marked feeds.

Key Words: Digesta • Flow • Markers • Models

	Introduction

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The mean residence time (MRT) of undigested residues in the gastrointestinal tract may be estimated by a compartmental model method (CMM) or by the indigestible entity (IE) pool dilution method (IEPD). The IEPD method estimates MRT as the time required for influx rate of IE to replace IE within a physically definable pool of digesta. The CMM method assumes a distribution of residence times resulting from specific mechanism(s) regulating IE flux within mixing pool(s) and nonmixing segments. With continued influx of IE, steady state residence time distributions of IE from a discrete meal cannot be distinguished by meal origin. A marker unique to a discrete meal and indelible to the IE of the discrete meal during its gastrointestinal sojourn is required to establish the sojourn time lapse of IE from the originating meal. The binding properties of rare earth elements suggest that they are appropriate flow markers of IE through less acidic segments of the gastrointestinal tract, such as the reticulorumen (rumen). However, evidence for indelibility of rare earths is indirect. The current report compares the MRT within the gastrointestinal tract (GMRT) of lambs of rare elements specifically applied to cottonseed hulls (CSH) vs the GMRT of indigestible fiber (PIF, as IE) contained within the CSH. Assumptions in estimating GMRT by CMM and IEPD are common except for the indelibility of the flux marker. Thus, statistically similar responses in GMRT for rare earth elements applied to CSH vs PIF within CSH would support the hypothesis that rare earths specifically applied to plant tissues are indelible markers of IE derived therefrom. To extend interpretations of indelibility beyond the CSH used here, two levels of dietary CP were used to elicit physiological responses specifically attributable to the nutrition of the rumen microbial ecosystem postulated as causal mechanisms regulating ruminal MRT.

	Materials and Methods

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General Experimental Approach.
This experiment was conducted following an Animal Use Protocol reviewed and approved by the Institutional Agricultural Animal Care and Use Committee of Texas A & M University. The study was the terminal phase of a larger study on the effects of CP sufficiency for ruminal digestion of NDF upon intake rate of NDF from CSH. Lambs had received the two diets of different CP levels in a crossover design for 30 d prior to the 19-d extra period of this experiment. Diets and feeding practices in the current extra period were the same as for the preceding 30-d with the addition of total fecal collections. Following an 8-d adjustment period, portions of the CSH in each meal for each lamb was replaced with rare earth-marked CSH for five consecutive days to effect a saturating phase of marker concentration in the feces during this continuous meal-feeding of rare earths. Feeding of rare earth-marked CSH was discontinued during the next 4 d to effect a desaturating phase of marker concentration in the feces. Lambs were then slaughtered after the last fecal collection in order to obtain weights and samples of digesta from segments of the digestive tract as required for calculating GMRT by the IDPD method. Feces collected during the 5-d saturating period and the 4-d desaturating phases were analyzed for concentrations of rare earth elements and used for estimating GMRT by the CMM method. The purpose for using this saturating and desaturating method was to minimize short-term meal-to-meal and longer-term effects of deviations in feed intake associated with ad libitum feeding.

Animals, Diets, and Feeding.
Five wether lambs (32.7 kg mean BW) were given ad libitum access to diets of either 52 or 123 g CP/kg DM during the 19 d. Animals were housed in an elevated stall permitting total collection of feces and urine, and an ambient environment was provided at 18 ± 3°C during a 12-h day–night cycle. Diets were either CSH or an 80:20 mixture of CSH plus cottonseed meal (CSH+CSM). Each diet was supplemented daily with 5 g/d of a mineral mixture containing (g/kg) 200 Ca, 55 P, 3 I, and 120 NaCl together with 352 IU vitamin D₂. The CSH analyzed (g/kg DM) 51.6 CP and 879 NDF, while the CSH+CSM contained 123 CP and 771 NDF. Diets were randomly assigned and fed as meals to individual animals twice daily (0600 and 1800) in amounts equal to 1.2 that disappearing during the corresponding 12-h interval of the previous day. During the preliminary period, refusals from previous meals were weighed and mixed with the assigned fresh diet and fed.

In order to ensure a complete and constant consumption of marked meals during the fecal collection period (d 10 through 14, 5 d), unconsumed diet (orts) was removed, and lambs were first offered partial meals of 35 g of the assigned diet formulated with CSH having specifically bound rare earth elements and solute marker. Consumption of the marked meal was complete within 1 h, after which additional unmarked diet was mixed with orts to provide an on-offer diet of 1.2 times that consumed during the corresponding 6-h period of the previous day. These 5 d constituted a marker-saturating phase of resident digesta prior to d 10. From d 15 through 18 (4 d), CSH treated identically to those marked but without added markers were fed to allow marker free CSH to dilute marker-bound CSH residues of meals prior to d 15 (i.e., a marker desaturating phase).

Animals were initially assigned to a staggered feeding schedule, such that one animal on each diet was slaughtered 2 h after the 0600 feeding on d 19. Subsequent to exsanguination, the gastrointestinal tract was excised and ligated at the esophageal orifice, the pyloric sphincter, the mid-jejunum, and the ileocecal fold. Digesta was removed from each site, weighed, and sampled for subsequent drying (60°C) and analysis.

Preparation of Marked Feed.
Rare earths were applied by soaking CSH overnight in a 0.1 M solution of acetic acid in amounts approximating their strong binding affinity (20 mg/g DM), and utilized 0.1 M acetic acid to remove rare earths bound to sites having affinities less than that of 0.1 M acetic acid (Ellis et al., 1994; Worley et al., 2002). Soluble salts of either nitrates, chlorides, or sulfates of La, Yb, and Tb were dissolved in 0.1 M acetic acid to provide approximately 1.3 mg of each element per milliliter of acetic acid, and each gram of CSH was soaked overnight in 5 mL of rare earth solution (20 mg of total rare earth element per gram CSH DM). After soaking for 12 h, excess solution was removed via filtration and the wet residue was soaked via occasional stirring in 5 mL of 0.1 M acetic acid per original gram of CSH for 1 to 2 h. Binding and washing in 0.1 M acetic acid were utilized to remove any loosely bound rare earth that would be displaced by a proton concentration equivalent to 0.1 M acetic acid. Subsequent to two washes with water to remove excess acetic acid and soluble rare earths, the rare earth-marked CSH were dried (60°C) and formulated into a diet for feeding. Upon feeding, 5 mL of 0.3 M cobalt diethylenetriaminopentaacetic acid was added as a solute marker to each 35-g portion of marked CSH.

Analysis.
Feed, fecal, and digesta samples were dried at 60°C, ground to pass through a 2-mm sieve, and stored for subsequent analysis. Total N was determined by the method of Munro and Fleck (1969). The NDF in feed and in in vitro residues was determined after 4 d incubation with rumen fluid (PIF, Lippke et al., 1986) using the procedures of Goering and Van Soest (1970). Neutron activation analysis of rare earths and Co was performed as described by Pond et al. 1985.

Estimation of Gastrointestinal Residence Time.
The G2O compartmental model (Ellis et al., 1994) was fitted to linked profiles of the saturating and desaturating phases of each rare earth and Co in the feces as described and illustrated by Wylie et al. (2000). This one-compartment, age-dependent model assumes a gamma-2 distribution of residence times (G2) and a discrete time delay () for undigested feed residues emerging in the feces (O). This one-compartment, age-dependent model was chosen because of the robustness of a one- vs two-compartment model and because the experimental objective was to determine the GMRT for comparison to GMRT estimated by the IEPD method. Least square estimation of the linked profiles of saturating and desaturating marker concentrations provided a sensitive procedure for estimating the mean turnover rate parameter (), and occurring over the total 10-d fecal collection period. The GMRT was computed as the sum of MRT in the age-dependent mixing compartment (2/) and the time delay (Ellis, et al., 1994).

The MRT within each gastrointestinal tract sampled was estimated by the IE pool dilution method utilizing PIF as the IE. Thus, MRT (h) = PIF (g) in the sampled anatomical segment at slaughter/mean hourly excretion rate of PIF observed via total fecal collections during the 6 d prior to slaughter. The GMRT within the total gastrointestinal tract was estimated as the sum of MRT in component segments of the total gastrointestinal tract. The mean age-independent turnover rate of PIF was computed as 1/MRT by the PIF pool dilution method.

Statistical Evaluations.
The GLM procedure of SAS (SAS Inst. Inc., Cary, NC) was used to determine significance of difference among parameters due to diet. Statistical differences among the three rare earth elements were evaluated as markers nested within animals. A paired t-test was used to determine differences between the two methods used to estimate MRT.

	Results and Discussion

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Means for parameters estimated by fitting the G2O model to fecal profiles for each rare earth element are summarized by diet in Table 1. Within diets, neither nor differed (P > 0.05) among the rare earth elements La, Yb, or Tb. Turnover rate parameters for the solute marker (cobalt ethylenetetraacetate) were approximately double that for the rare earth elements (0.048 vs 0.025/h; P < 0.001), while the time delay did not differ (P = 0.71) among the three rare earth elements but did differ for rare earth elements vs Co (P > 0.001). The for individual rare earth elements and for Co was approximately twofold (P < 0.01) greater in lambs consuming the CSH+CSM vs CSH diet for rare earth elements (0.024 to 0.026 h vs 0.043 to 0.052 h) and for Co (0.048 vs 0.097 h). While differed via diet, diet had no effect (P > 0.05) upon .

View this table: [in this window] [in a new window]   Table 1. Means for gamma-2 age-dependent rate parameter, (), and time delay, (), estimated from fecal profiles of rare earth marker elements (La, Yb, and Tb) applied to cottonseed hulls (CSH), and for a solute Co in lambs receiving CSH or CSH plus cottonseed meal (CSH+CSM) diets

View larger version (14K): [in this window] [in a new window]   Figure 1. Relationship between gastrointestinal mean residence time (GMRT) of rare earth elements and potentially indigestible fiber (PIF) in cottonseed hulls (+) and cottonseed hull plus cottonseed meal () diets.

The criteria of similar responses in GMRT for rare earths vs PIF is a critical one in that the responses in GMRT reflect a wide range in physiological responses of interest in ruminant nutrition, nutritional effects upon the rumen microbial ecosystem, and consequential turnover of undigested feed residues. Presumably, variations in GMRT were the result of nutritional responses by the ruminal microbial ecosystem to sufficiency of CP in supporting the increased growth rate of NDF-digesting rumen bacteria, and thereby increased the intake rate of potentially digestible NDF in response to a flux of metabolizable AA (Ellis et al., 1999; Ellis and Matis, 2001; and Lowe et al., 2001).

Segmental MRT.
Estimates of MRT via the IEPD method in specific anatomical segments of digesta are summarized in Table 3 by diet. Overcoming a CP insufficiency in the rumen (CSH+CSM vs CSH diets) resulted in smaller MRT (P < 0.05) in gastric segments (reticulorumen, the omasal body, and the abomasum) and in the cecum and the distal colon-rectum, but had no effect (P < 0.05) on MRT in intestinal segments (proximal and distal small intestines and the proximal colon). The MRT in preduodenal sites represented 0.83 and 0.84 of the GMRT in the total tract for diets CSH+CSM and CSH diets, respectively (Table 3). The regression of CMRT due to mixing flow within the gastrointestinal tract estimated by CMM (2/; Table 3) was regressed against GMRT determined by the IEPD method and yielded a regression coefficient of 0.83 ± 0.016. Proportions of CMRT/GMRT observed here are essentially equivalent to proportions in the order of 0.85 reported by Wylie et al. (2000) for a forage diet fed to cattle.

We propose that the proportions of GMRT due to mixing flow in the rumen/mixing flow in the total gastrointestinal tract is relatively constant (in the order of 0.9 for forage diets fed to ruminants), and at least for forage fed ruminants, could be predicted from fecal profiles of rare earth markers. Indeed, rumen MRT from GMRT may be more accurately predicted from GMRT in view of the inability to representatively sample ruminal flow paths within ruminal digesta (Ellis et al., 1994) and problems of obtaining representative samples from cannulated postruminal sites.

Time Delay.
The mean estimated by CMM were 9.7 and 9.3 h for lambs fed the CSH and CSH+CSM diets, respectively (Table 3). These values are of a same magnitude as those summarized from the literature regarding sheep and cattle fed a variety of diets (Ellis et al., 1994, 1999; Wylie et al., 2000). Similar time delays across diets and animals suggest that time delays for nonmixing flow in cattle and sheep are relatively constant and independent of extreme differences in ruminal CMRT, such as those observed in the current results.

A discrete time delay is estimated by CMM as the transit time for first appearance of a detectable marker between the dose and sampling site. In the absence of mixing flow, such transit time would be represented by a "spike" recovery of marker dose on first appearance indicating perfect lamina flow. Conceptually, lamina flow would be expected in anatomical segments having a small bore lumen without taenia and/or haustria (hindgut structures opposing laminar flow; Langer, 1984), and in digesta segments having viscous, low-moisture digesta resistant to mixing (distal colon and rectum). Using such criteria, no combination of anatomical segments sampled in the present experiment (Table 3) yielded summated MRT approximating the magnitude of time estimated by CMM as a discrete time delay.

We suggest that CMRT in the postruminal segments is derived from numerous flow paths of digesta in contrast to simple mixing vs discrete time delay of a lamina flow process frequently used in deterministic exponential based CMM. Use of age-dependent residence time distributions as used here partitions more of a time delay into CMRT (see Figures 5 and 6C of Pond et al., 1988). It is this nonlaminar "mixing" flow through postruminal segments of the gastrointestinal tract that accounts for the relatively constant fraction of observed GMRT being attributed to the postruminal MRT (0.84). In summary, specific digesta sites contributing to time delay as usually estimated by the CMM method remain elusive.

Rare Earth Binding.
Published conclusions that rare earth elements migrated from feed residues to which they were initially applied are probably the result of one or more of the following: 1) applying rare earths in excess of their strong binding capacity, 2) failure to remove excess and unbound rare earths, and 3) assumptions involving rate of reduction in size of feed residues and their size relative to porosity in situ bags used (Hartnell and Satter, 1979 and Owens and Hanson, 1992). Clearly, rare earth elements are disassociated by proton concentrations at a pH less than 4 (Worley et al., 2002). Conceptually, some migration may occur if the rare earth-entity binding site is disrupted. However, it appears probable that entities having stronger binding sites for rare earths are either intrinsically resistant to digestion or the specific binding site is resistant to digestion (Worley et al., 2002). If rare earth binding affects digestion attributes, then these effects should be lessened by minimizing the level of rare earth binding, manipulating meal size doses, and utilizing the most sensitive methods of rare earth analysis. Strong binding affinities for rare earths appear positively related to phenolic compounds and to less digestible entities of NDF (Allen et al., 1985), and range up to 20 mg element per gram of feed residues. Thus, critical use of rare earths as flow markers necessitates concentrations of rare earths generally less than 5 mg/g DM and correspondingly large doses of marker feed and sensitive methods for rare earth analysis in order to reliably detect rare earth elements remaining after several orders of dilution turnover.

	Implications

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Estimates of flow parameters in the reticulorumen and the gastointestinal tract of ruminants can be estimated from compartmental models fitted to profiles of rare earth elements initially applied specifically to fibrous feeds.

	Footnotes

 
² Deceased.

Received for publication January 30, 2002. Accepted for publication June 19, 2002.

	Literature Cited

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