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Omasal Flow of Soluble Proteins, Peptides, and Free Amino Acids in Dairy Cows Fed Diets Supplemented with Proteins of Varying Ruminal Degradabilities¹

S. M. Reynal^*,2,3, I. R. Ipharraguerre^,4, M. Liñeiro^,5, A. F. Brito^*,6, G. A. Broderick and J. H. Clark

^* Department of Dairy Science, University of Wisconsin, Madison 53706
Department of Animal Sciences, University of Illinois, Urbana 61801
Agricultural Research Service, USDA, US Dairy Forage Research Center, University of Wisconsin, Madison 53706

² Corresponding author: sreynal{at}wisc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Three ruminally and duodenally cannulated cows were assigned to an incomplete 4 x 4 Latin square with four 14-d periods and were fed diets supplemented with urea, solvent soybean meal, xylose-treated soybean meal (XSBM), or corn gluten meal to study the effects of crude protein source on omasal canal flows of soluble AA. Soluble AA in omasal digesta were fractionated by ultrafiltration into soluble proteins greater than 10 kDa (10K), oligopeptides between 3 and 10 kDa (3–10K), peptides smaller than 3 kDa (small peptides), and free AA (FAA). Omasal flow of total soluble AA ranged from 254 to 377 g/d and accounted for 9.2 to 15.9% of total AA flow. Averaged across diets, flows of AA in 10K, 3–10K, small peptides, and FAA were 29, 217, 50, and 5 g/d, respectively, and accounted for 10.3, 71.0, 17.5, and 1.6% of the total soluble AA flow. Cows with diets supplemented with solvent soybean meal had higher flows of Met, Val, and total AA associated with small peptides than those whose diets were supplemented with XSBM, whereas supplementation with corn gluten meal resulted in higher total small peptide-AA flows than did XSBM. Averaged across diets, 27, 75, and 93% of soluble AA in 10K, 3–10K, and peptides plus FAA flowing out of the rumen were of dietary origin. On average, 10% of the total AA flow from the rumen was soluble AA from dietary origin, indicating a substantial escape of dietary soluble N from ruminal degradation. Omasal concentrations and flows of soluble small peptides isolated by ultrafiltration were substantially smaller than most published ruminal small peptide concentrations and outflows measured in acid-deproteinized supernatants of digesta.

Key Words: dairy cow • omasal flow • soluble amino acid • peptide

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Protein degradation by ruminal microbes results in the formation of ammonia, and peptides and AA are intermediates in this process. Ammonia that accumulates in excess of microbial requirements is converted to urea in the liver and excreted in the urine, resulting in inefficient N utilization by the animal (Broderick et al., 1991). In vitro and in vivo evidence suggests that the rate-limiting step in ruminal protein degradation may be peptide hydrolysis (Russell et al., 1983; Chen et al., 1987b), and the degradation rate may depend on the degradability (Broderick and Wallace, 1988) and AA profile of the protein (Yang and Russell, 1992). Mounting evidence indicates that peptides and free AA (FAA) stimulate microbial yield and fermentation (Argyle and Baldwin, 1989; Chikunya et al., 1996; Carro et al., 1999). Furthermore, peptides (mainly diand tripeptides) may contribute significantly to AA absorption from the intestines of ruminants (Remond et al., 2000) and may have nutritional benefits for the host animal (Webb and Matthews, 1998). Therefore, alteration of proteolysis and peptide formation in the rumen by dietary manipulation may have nutritional benefits for both ruminal microbes and the host animal, improving utilization of dietary N for productive purposes.

New systems of ruminant ration formulation (AFRC, 1992; Sniffen et al., 1992; NRC, 2001) rely on rate constants for ruminal protein degradation and passage. Most commonly, the in situ method has been used to assess the kinetics of protein degradation in the rumen. However, this technique has theoretical limitations that call its accuracy into question (Broderick et al., 1991). Two major concerns are 1) that the rates of ruminal degradation of the soluble N fraction (fraction A) from all feeds incubated in situ are assumed to be infinite and 2) that the portion of dietary protein that is degraded in the rumen (fraction B) is assumed to be entirely used for microbial protein synthesis or production of ammonia and carbon skeletons, or both. However, if a portion of these fractions (i.e., A and B) escapes ruminal degradation as soluble proteins, peptides, and free AA, the validity of these assumptions may not hold true.

Despite the potential nutritional and metabolic importance of the ruminal peptide pool, most of the information available on degradation and metabolism of soluble N fractions in ruminants is based on methodologies that may be unreliable. Peptide AA are usually determined by quantitation of free AA concentrations before and after hydrolysis of samples previously deproteinized by acid precipitation. However, a substantial proportion of the soluble proteins may remain in solution after acid deproteinization (Moughan et al., 1990) and the use of strong acids may result in partial hydrolysis of proteins and release of peptides, leading to an overestimation of the peptide N fraction (Seal and Parker, 1998; Choi et al., 2002a). Moreover, the extent of this chemical deproteinization depends on the type and concentration of the acid used (Greenberg and Shipe, 1979; Moughan et al., 1990; Butts et al., 1991) and on the structure of the proteins being precipitated (Bhatty, 1972; Greenberg and Shipe, 1979). Because the acid type (e.g., perchloric, trichloroacetic, sulfosalicylic), concentration, and times and temperatures of incubation vary widely among published methodologies, interpretation and comparison of data obtained using these techniques should be done with caution. Therefore, a more reliable and reproducible method is needed to study the nutritional and metabolic significance of the flow of soluble AA from the rumen and to test the validity of the in situ technique for estimating rates of protein degradation in the rumen.

The objective of this experiment was to study the effect of feeding diets differing in concentration, ruminal degradability, and source of protein supplement on flows of soluble proteins, peptides, and FAA at the omasal canal with special emphasis on the methodology used for soluble N fractionation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Animal Management, Experimental Design, and Sampling
Three ruminally and duodenally cannulated lactating Holstein cows averaging 154 (±92) DIM at the beginning of the trial were assigned to an incomplete 4 x 4 Latin square with four 14-d periods and were fed one of 4 diets differing in the proportion and source of the protein supplement. Each cow was randomly assigned to 1 of 3 treatment sequences in which a treatment never followed the same treatment more than once. The TMR contained (DM basis) 35% corn silage, 25% alfalfa silage, 2.7% vitamins and minerals, and 37% concentrate [CP supplement plus ground shelled corn (GSC)]. Diets (Table 1) differed in the proportion and source of CP supplement: diet A (urea; 34.8% GSC, 2.3% urea); diet B [32.1% GSC, 5.2% solvent soybean meal (SSBM)]; diet C [32.7% GSC, 4.6% xylose-treated SBM (XSBM; SoyPass, LignoTech USA, Inc., Rothschild, WI)]; or diet D [28.6% GSC, 8.5% corn gluten meal (CGM)]. Details of the surgical procedures, animal management, and sampling and analyses of silages, concentrates, TMR, and orts are described by Ipharraguerre et al. (2007).

Digesta flow leaving the rumen was quantified using the omasal sampling technique developed by Huhtanen et al. (1997) as modified by Ahvenjärvi et al. (2000). Indigestible NDF, CoEDTA, and YbCl₃ were used as markers to assess digesta flows at the omasal canal, and the external microbial marker ¹⁵N was used to measure microbial N flows from the rumen. Details on marker preparation, infusion, and analyses are reported in Ipharraguerre et al. (2007). Omasal spot samples of 355 mL were collected from the omasal canal on d 12, 13, and 14 of each period such that the 12 samples represented a 24-h feeding cycle over 3 d. Each spot sample was swirled vigorously and poured into 3 containers to obtain subsamples of 200, 125, and 30 mL. The 125-mL subsamples were pooled over the 4 daily sampling times to yield one 500-mL composite from each cow on each sampling day, and the composite sample was processed later that day to isolate bacteria. The 200-mL subsamples were pooled and stored at –20°C as they were collected over all 12 sampling times, to yield one 2.4-L omasal composite from each cow in each period. The 30-mL subsamples were squeezed through 1 layer of 0.45-µm Dacron mesh, the filtrate centrifuged at 50,000 x g for 15 min at 5°C to precipitate particulate matter, and the supernatant was held on ice until stored at –80°C within 30 min of collection for later determination of soluble N flow at the omasal canal.

The 2.4-L pooled omasal composites were separated into 3 digesta phases: fluid phase (FP), large particle phase (LP), and small particle phase (SP). Concentrations of Co, Yb, and indigestible NDF in LP and SP and of Co and Yb in FP were used to mix DM from freeze-dried FP, SP, and LP in the correct proportions to reconstitute the omasal true digesta (OTD) flowing out of the rumen based on the triple-marker method of France and Siddons (1986). Digesta phase separation and marker analyses are described in Ipharraguerre et al. (2007). Aliquots of the SP and LP phases were mixed in the correct proportions based on the markers to yield a 2-g sample that was ground through a 0.5-mm screen with a Udy Cyclone Sample mill (Udy Corporation, Fort Collins, CO) and defined as small plus large particles (SP + LP). Feed and OTD samples were sequentially ground through 2- and 1-mm screens using a Wiley mill (Arthur H. Thomas, Philadelphia, PA) and analyzed for DM at 105°C; ash and OM (AOAC, 1980); and total N using a combustion assay (Leco FP-2000 N Analyzer; Leco Instruments, Inc., St. Joseph, MI).

At the end of each sampling day, the 500-mL omasal composites were separated to yield fluid and particle phases that were equivalent to the FP and the SP + LP phases from the 2.4-L composites, respectively. Fluid-associated bacteria (FAB) and particle-associated bacteria (PAB) were isolated from these fluid and particle phases, respectively, and pooled over the 3 sampling days of each period to yield one composite each of FAB and PAB per cow per period. The OTD, FAB, PAB, and individual feeds were analyzed for 16 AA after hydrolysis in 6 N HCl (Nagel and Broderick, 1992), using norleucine as the internal standard and ion-exchange chromatography with ninhydrin detection (Beckman 6300 Amino Acid Analyzer; Beckman Instruments, Inc., Palo Alto, CA). Samples of ¹⁵N natural background, FAB, PAB, FP, and SP + LP (100 µg of N) were weighed into tin cups (5 x 9 mm; Costech Analytical Technologies, Valencia, CA) and, after addition of 50 µL of 72-mM K₂CO₃ solution, were placed in a 60°C oven to volatilize ammonia. Samples were analyzed for NAN and ¹⁵N enrichment of NAN by isotope ratio mass spectrometry (Stable Isotope Facility, Department of Agronomy and Range Science, University of California-Davis, Davis, CA). Other details about collection, processing, and analysis of samples of OTD, FAB, and PAB are reported in Ipharraguerre et al. (2007).

Fractionation of Soluble N Fractions
At the end of the experiment, the 30-mL omasal subsamples were fractionated as outlined in Figure 1. Samples were slowly thawed in an ice bath and a 13.5-mL aliquot was centrifuged at 50,000 x g for 15 min at 0°C. The supernatant was filtered through a 0.1-µm filter (Millex-VV, no. SLVV033RB, PVDF filter, Millipore, Billerica, MA) and 5 mL of filtrate was saved for later analysis of FAA. A 5.4-mL aliquot of filtrate was pipetted into a tared 10-kDa molecular weight (MW) cut-off ultracentrifugation filter with a cellulose membrane (Amicon-Ultra, no. UFC901024, Millipore) and 0.6 mL of 2 mM homoarginine solution was added as an internal standard for determination of FAA in retentates. This filtration device was centrifuged at 5,000 x g for 30 min at 5°C. Then, 6 mL of McDougall’s buffer (McDougall, 1948) was added to the retentate and the device was centrifuged again at 5,000 x g for 30 min at 5°C. The vials from the filtration device containing filtrate and retentate solutions were weighed and their weights recorded for recovery calculations. A 0.5-mL aliquot of retentate was transferred to a glass culture tube for later hydrolysis and determination of AA in soluble proteins greater than 10 kDa (10K) in omasal digesta. The remaining retentate solution was stored at –80°C for later analysis. An 8-mL aliquot of filtrate was transferred to a tared 3-kDa MW cut-off ultracentrifugation filter with a cellulose membrane (Centriprep-3, no. 4302, Millipore) and centrifuged once at 3,000 x g at 5°C for 65 min and then 4 more times for 20 min at the same speed and temperature. Filtrate and retentate vials containing their corresponding solutions were weighed and their weights recorded for recovery calculations. A 0.25-mL aliquot of retentate and a 1-mL aliquot of filtrate were transferred to glass culture tubes for later hydrolysis and determination of individual AA in soluble oligopeptides between 3 and 10 kDa (3–10K), and peptides of less than 3 kDa (called small peptides hereafter) plus FAA. The remaining retentates and filtrates were stored at –80°C for later analysis. To the tubes containing 0.5 mL of 10K retentate and 0.5 mL of small peptides plus FAA filtrate, 0.625 mL of 12 N HCl with 1 g/L of phenol and 0.125 mL of 1 mM norleucine were added. To tubes containing 0.25 mL of 3–10K retentate, 312.5 µL of 12 N HCl with 1 g/L of phenol and 62.5 µL of 1 mM norleucine were added. All tubes were flushed with N₂ gas for 20 s, sealed with Teflon tape and capped tightly, and heated at 110°C for 24 h (Gehrke et al., 1985; Nagel and Broderick, 1992). Hydrolysates were evaporated to moistness using a concentrator (SpeedVac Concentrator SVC-200H, Savant Instruments, Inc., Farmingdale, NY), redissolved in 1 mL of distilled water, evaporated again, dissolved in 1 mL of pH 2.2 sodium citrate buffer (Na-S, Beckman Instruments, Inc., Palo Alto, CA), and analyzed for individual AA by ion-exchange chromatography as described earlier. The 5-mL filtrates saved for FAA analysis were evaporated to moistness and dissolved in 2 mL of pH 2.2 sodium citrate buffer. An attempt was made to analyze FAA on this nonhydrolyzed sample containing peptide- and protein-bound AA. However, ammonia and peptide-bound AA appeared to saturate the binding sites of the ion-exchange column, hindering chromatographic separation. Therefore, 1 mL of 2 M K₂CO₃ was added to 1 mL of evaporated and buffer-dissolved sample, heated at 60°C for 24 h to volatilize ammonia, the pH adjusted to 2.2 by adding 2 mL of 0.16 M sodium citrate and 2.2 M HCl, and filtered through a 3-kDa MW cut-off ultracentrifugation filter before analysis of individual AA by ion-exchange chromatography as described earlier. The concentration of homoarginine in each of the 4 soluble N fractions was used to compute the content of each AA in its free form in the 10K, 3–10K, and small peptide fractions as follows:

View larger version (29K): [in this window] [in a new window]   Figure 1. Fractionation and analysis of soluble proteins, peptides, and free AA in omasal digesta. PVDF = polyvinylidene fluoride; HArg = homoarginine (internal standard); MW = molecular weight; 3–10K = 3 to 10 kDa; 10K = 10 kDa.

where bound AA fraction is either 10K, 3–10K, or small peptide.

Free AA remaining in bound AA fractions were subtracted from those fractions and added to the FAA fraction. Remaining retentates from the 10-kDa membrane and both retentates and filtrates from the 3-kDa membranes were thawed and an equal volume of 2 M K₂CO₃ was added. Samples were evaporated using a SpeedVac concentrator to a volume of approximately 200 µL. Concentrated samples were transferred to tin cups (5 x 9 mm; Costech Analytical Technologies, Valencia, CA) and placed in a 60°C oven for 24 h to volatilize ammonia. Samples were analyzed for ¹⁵N enrichment by isotope ratio mass spectrometry as described earlier. The ¹⁵N enrichments of FAB and PAB and of each soluble N fraction were used to calculate the proportion of soluble N of microbial origin in each fraction.

Statistical Analyses
Data were analyzed using PROC MIXED in SAS (SAS Institute, 1999). The following model was fitted to all variables that did not have repeated measurements over time:

where Y_jkl is the dependent variable, µis the overall mean, P_j is the effect of period j, C_k is the effect of cow k, T_l is the effect of treatment l, and _jkl is the residual error. The degrees of freedom for treatment were partitioned into 3 single-degree-of-freedom orthogonal contrasts: urea vs. true protein (urea vs. CGM), high vs. low predicted protein degradability (SSBM vs. XSBM), and soybean vs. corn protein (XSBM vs. CGM). All terms were considered fixed except for C_k, which was considered random.

The following model was used for ruminal variables for which there were repeated measurements over time (pH, NH₃, and total FAA):

where Y_jklm is the dependent variable, µis the overall mean, P_j is the effect of period j, C_k is the effect of cow k, T_l is the effect of treatment l, _jkl is the whole plot error, Z_m is the effect of time m, ZT_ml is the interaction between time m and treatment l, and _jklm is the sub-plot error. Based on the largest value for Akaike’s information criterion (Littell et al., 1996), the heterogeneous autoregressive structure ARH(1) was selected as the appropriate covariance structure. The subject of the repeated measurements was defined as cow (square x period x treatment). All terms were considered fixed, except for C_k, which was considered random. Because the ZT_ml interaction was not significant at a = 0.05 for any of the ruminal variables measured, treatment means were compared across sampling times using the contrasts described above. Differences between least squares means were considered significant at P < 0.05, and differences were considered to indicate a trend toward significance at 0.05 < P < 0.10.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Diet Composition, Animal Production, and Ruminal Digestion
Diets were formulated to be isoenergetic (mean = 1.58 Mcal/kg of NE_L) and to provide 2 levels of CP: high (19.7 and 18.2% for diets A and D) and low (15.8 and 15.3% for diets B and C; Table 1). Within each CP level, diets differed in the N form (urea vs. true protein for diets A and D), the predicted ruminal degradability of the soybean protein (nontreated vs. treated SBM for diets B and C), and the source of the protein (soybean vs. corn for diets C and D). Dietary concentrations (% of dietary DM) of NDF, ADF, starch, and NFC ranged from 28.1 to 31.8%, from 17.5 to 20%, from 26.1 to 28.1%, and from 41.2 to 45.5%, respectively. Diets differed substantially in the proportion of the total N in AA, with diet D being highest (82.3%), diets B and C intermediate (68.8 and 64.4%, respectively), and diet A the lowest (35.5%). The chemical composition of the silages and concentrate mixes is presented in Table 2.

When fed high-CP diets, cows whose diets were supplemented with urea had similar DMI, ruminal pH, and FAA concentrations but a higher ruminal ammonia concentration (Table 3) than cows whose diets were supplemented with CGM. Previous reports indicate that intakes of DM by lactating cows fed diets supplemented with urea were depressed, either significantly (Reynal and Broderick, 2003) or not significantly (Wohlt et al., 1991), by 2 kg/d compared with cows whose diets were supplemented with CGM. However, urea-supplemented diets contained less CP than did CGM diets, confounding the effects of source and level of dietary protein (Wohlt et al., 1991; Reynal and Broderick, 2003). Within the low-CP diets, there was no difference between SSBM and XSBM on DMI or any of the ruminal variables measured. Replacing XSBM with CGM resulted in a significant decrease in DMI and ruminal FAA concentration. These results are in agreement with data from a meta-analytic review of the literature (Ipharraguerre and Clark, 2005) showing that the replacement of SSBM with corn by-products frequently depresses DMI by lactating dairy cows. It should be noted, however, that in this study the effects of dietary protein source (soybean vs. corn) and concentration (15.3 vs. 18.2% CP) are confounded. Within CP level, neither urea vs. true protein nor SSBM vs. XSBM had significant effects on omasal flows and ruminal digestibilities of DM and OM (Table 3). Higher DMI for cows fed XSBM resulted in higher flows of DM and OM at the omasal canal, compared with cows fed CGM. However, apparent ruminal digestibility of DM and true ruminal OM digestibility were not affected by dietary treatment. As expected, cows fed diets supplemented with urea had smaller flows of total NAN and nonammonia nonmicrobial N (NANMN) at the omasal canal but a higher true ruminal digestibility of N than cows fed CGM (Table 4). Cows fed XSBM had a greater flow of NANMN (222 vs. 177 g of N/d) but a similar flow of microbial NAN compared with cows fed SSBM, resulting in a trend (P < 0.09) for a greater flow of NAN. Previous work showed similar changes in the ruminal outflow of N fractions when SSBM was replaced with XSBM (Ipharraguerre et al., 2005) and other rumen-protected soy products (Ipharraguerre and Clark, 2005). Replacing XSBM with CGM depressed the omasal flows of total NAN (from 622 to 538 g/d) and NANMN (from 222 to 163 g/d), at least in part because of the associated effects on DMI. Microbial efficiencies were not significantly affected by dietary treatments (Table 4).

The cut-offs of tungstic and trichloroacetic acids are considered to be approximately at peptide sizes of 3 and 10 AA, respectively (Licitra et al., 1996). Assuming an average MW of 127 per AA (based on the average occurrence of individual AA in over 200 proteins; Klapper, 1977), 3 and 10 AA would correspond to average MW of 381 and 1,270, respectively. However, the extent of deproteinization and the MW of the proteins being precipitated are dependent on the type and concentration of the acid used. Trichloroacetic (10% wt/ vol) and perchloric acids (7% wt/vol) precipitated, respectively, 26 and 70% of the total soluble N in ileal digesta of rats (Butts et al., 1991). Greenberg and Shipe (1979) reported that complete precipitation of ß-LG was achieved at about 15, 20, and 0.5% (wt/vol) of trichloroacetic, sulfosalicylic, and tungstic acids, respectively. In addition, more than one-half of the soluble N from ileal digesta of rats remained in solution after deproteinization with perchloric (7% wt/vol) and trichloroacetic acids (10% wt/vol) with, respectively, 35 and 69% of the supernatant N being in the form of soluble protein (Ohara and Ariyoshi, 1979; Moughan et al., 1990). Additionally, the extent of precipitation also depends on protein structure. When TCA was used at 5% (wt/vol) to precipitate ß-LG, BSA, keratin, hemoglobulin, myosin, and actomyosin, 12, 0, 14, 4, 1, and 21% of the protein remained in the supernatant, respectively (Greenberg and Shipe, 1979). Even at a 10% (wt/vol) concentration of TCA, 11% of keratin remained in solution (Greenberg and Shipe, 1979). In the study of Bhatty (1972), complete precipitation of soluble proteins from rapeseed meal required a 6-fold increase in TCA concentration compared with that required to precipitate CN and hemoglobin. Furthermore, as suggested by Choi et al. (2002a) and Seal and Parker (1998), the use of strong acids may result in partial hydrolysis of proteins, release of peptides, and overestimation of the peptide-N fraction. Therefore, interpretation and comparison of data obtained using these techniques should be done with caution.

Alternatively, soluble N fractions can be separated using UF with membranes of different MW cut-offs without addition of acids. When filtered through a 10-kDa cut-off membrane, from 88 to 94% (mean 91%) of the N in purified proteins of MW between 14,300 and 669,000 Da was recovered in the retentate, whereas from 90 to 100% (mean 95%) of the N in peptides between 146 and 6,000 Da was recovered in the filtrate solution (Butts et al., 1991). When peptide concentrations in sheep blood and plasma measured in the filtrate from a 3-kDa cut-off filter were compared with those measured in the supernatant after deproteinization with sulfosalicylic acid, chemical deproteinization overestimated peptide concentrations in blood and plasma samples by 160 and 56%, respectively (Bernard et al., 2001). Backwell (1998) reported that oligopeptides of MW as high as 9.3 kDa remained in the supernatant after deproteinization of sheep blood samples with sulfosalicylic acid (final concentration of 13% wt/ vol). When plasma samples from sheep that were deproteinized with sulfosalicylic acid (final acid concentration of 3.3% wt/vol) were filtered through a 3-kDa cut-off membrane, peptide AA concentrations were less than half of those in deproteinized samples without filtration (Remond et al., 2000). Ultrafiltration has also been used as a pretreatment before separating peptide AA by reverse-phase chromatography (Seal and Parker, 1996). Because UF, alone or followed by chromatographic separation, should not affect the structure and solubility of proteins and peptides, its use may result in more accurate estimates of soluble N fractions compared with chemical deproteinization. However, the selectivity of the membranes used in UF depends on the size and shape of the proteins and decreases as the MW approaches the cut-off value of the membrane (Butts et al., 1991; Millipore Technical Guide, Millipore Corp.). Moreover, a small proportion of the total volume filtered containing molecules smaller than the MW cut-off will remain in the retentate. Therefore, the MW cut-offs of the membranes are approximate values and some degree of cross-contamination among fractions should be expected. Caution is needed when handling the samples during collection and analysis to avoid protein degradation and contamination. Bacterial and protozoal cells should be immediately removed after sampling to avoid cell lysis and overestimation of soluble N fractions. Functional proteases and peptidases will result in inaccurate estimates of soluble N fractions if supernatants are not immediately processed or stored, preferably at –80°C, until processed. These are some disadvantages compared with chemical deproteinization, where enzyme activity is immediately stopped by acidification, facilitating sample handling. Nonetheless, immediate high-speed centrifugation followed by freezing in liquid N should minimize degradation and contamination of soluble N fractions separated by UF. Therefore, UF of properly processed and stored samples is a preferred method of separating soluble N fractions than chemical deproteinization.

Omasal Flows of Total AA in Each Soluble N Fraction
Omasal flows of AA in proteins greater than 10K averaged 29 g/d across diets (P > 0.10), accounting for 1.1 to 1.6% of total AA flow and from 7 to 13% of the total soluble AA flow (Table 9). Flows of AA in soluble 3–10K oligopeptides averaged 217 g/d across diets (P > 0.10) and accounted for 6.4 to 12.5% of total AA flows at the omasal canal and for most of the soluble AA flow (65 to 79%; Table 10). Cows supplemented with urea tended (P = 0.09) to have lower proportions of soluble AA as 3–10K oligopeptides than cows fed true protein from CGM (65.4 vs. 79.3%). Soluble AA fractions containing bound AA of MW above 3 kDa accounted for 78 to 86% of the soluble AA flow and 7.5 to 13.6% of the total AA flow at the omasal canal. On the contrary, soluble protein (defined as NAN in a 10% TCA precipitate) accounted for only 0.9 to 1.6% (Choi et al., 2002b), 21 to 23% (Choi et al., 2003), and 20 to 26% (Choi et al., 2002a) of the total omasal flow of soluble NAN (SNAN). However, incomplete precipitation of proteins by acid deproteinization might have resulted in underestimation of soluble proteins in these studies.

Omasal Flows of Individual AA Associated with Each Soluble N Fraction
Only 7 out of 18 AA assayed by ion-exchange chromatography (Arg, His, Ile, Leu, Lys, Phe, and Tyr) were found at detectable concentrations in their free form (Table 12). Concentrations of total FAA (sum of 7) in omasal digesta averaged 26 mg of AA/L across diets. Flows of individual AA with soluble proteins greater than 10K were not significantly affected by dietary treatment (Table 9). When compared with urea, supplementation with CGM resulted in higher flows of His (8.5 vs. 5.5 g of AA/d) and a trend for significantly higher flows of Ile (P < 0.10) and Val (P < 0.10) associated with soluble oligopeptides of 3–10K (Table 10). Cows fed CGM tended (P < 0.10) to have higher flows of His and Val in 3–10K oligopeptides than cows fed XSBM.

There was no difference between urea and CGM or XSBM and CGM on flows of individual small peptide-AA (Table 11). However, flows of Met, Val, Ala, Cys, Gly, and total AA in peptides of less than 3 kDa were significantly higher in cows fed SSBM than XSBM. Yang and Russell (1992) reported that dipeptides containing Pro and either Met, Gly, or Val were degraded by ruminal bacteria in vitro at rates that were more than 2-fold slower than those of dipeptides containing Met, Gly, and Val in combination with AA other than Pro. Although in the present study peptide-bound Pro was not significantly affected by diet, flows of small peptide-bound Met, Gly, Val, and total AA were 460, 25, 44, and 29% higher, respectively, in cows fed SSBM than XSBM. Therefore, feeding SSBM might have resulted in greater formation and accumulation of peptides containing Pro in combination with Met, Gly, and Val, compared with XSBM. Similarly, passage of ruminal peptides (determined in the chemically deproteinized supernatant of rumen fluid) increased with increasing intakes of degradable protein by cows in diets supplemented with either SSBM, extruded SBM, or fish meal (Chen et al., 1987b).

Flows of Ile and Lys in their free form were significantly higher for cows whose diets were supplemented with urea, compared with CGM (Table 12). Cows fed SSBM had higher flows of Ile (P = 0.02) and a trend for significantly higher flows of His, Lys, and Phe compared with cows whose diets were supplemented with XSBM.

Despite the alteration of the individual soluble AA flow from the rumen of cows by dietary manipulation observed in the present study, the extent of these changes was relatively small compared with the total flow of each AA. Although the flow of Met in small peptides was 5.6 times higher for cows fed SSBM than for cows fed XSBM (1.12 vs. 0.2 g/d; Table 11), the 0.92 g/d difference represented only 1.3% of the total flow of Met at the omasal canal (mean 70 g/d; Table 6).

Omasal Flows of Soluble AA from Microbial and Dietary Origin
The microbial contribution to total SNAN flows at the omasal canal of cows fed diets containing grass silage, barley, and rapeseed meal averaged 61 (Choi et al., 2002a) and 71% (Choi et al., 2003) across diets. However, microbial SNAN flows were estimated based on ¹⁵N enrichment of the supernatant of omasal samples that were acidified with H₂SO₄ before centrifugation. The addition of a strong acid to digesta samples before removal of microbial cells by centrifugation could have resulted in cell lysis, release of cytoplasmic soluble N, and overestimation of microbial contributions to SNAN flows. On the contrary, in the present study only 25% of oligopeptides and 7% of peptides plus FAA flowing at the omasal canal were of microbial origin (Tables 10–12). Although most of the soluble proteins greater than 10K were of microbial origin (mean 73%), this fraction constituted only 10% of the total soluble AA flowing at the omasal canal (Table 9). Dietary soluble AA contributed, on average, 73% of total soluble AA and 10% of total AA flows (Table 13), indicating a substantial escape of dietary soluble AA from ruminal degradation. These results call into question the validity of assuming that ruminal degradation of the soluble N fraction (fraction A) of feeds incubated in situ occurs at an infinite rate and that the portion of protein that is degraded in the rumen (fraction B) is solely used for microbial protein synthesis, production of ammonia and carbon skeletons, or both.

Soluble AA flows of dietary origin were significantly affected by dietary treatment. Replacing XSBM with CGM resulted in higher flows of total (206 vs. 271 g/ d) and oligopeptide-associated (151 vs. 216 g/d) soluble AA of dietary origin, whereas replacing urea with CGM increased oligopeptide-associated (110 vs. 216 g/d) and total (168 vs. 271 g/d) dietary soluble AA flows (Tables 10 and 13). The flow of soluble peptides of dietary origin in cows fed SSBM was significantly higher than for cows fed XSBM (57 vs. 43 g/d; Table 11). Differences in DMI among treatments might have accounted for part of these findings.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
The study of the metabolic and nutritional significance of ruminal peptide and FAA pools to both microbes and the host animal depend on accurate and reproducible techniques for isolating and analyzing these fractions. Based on data reported in this study and in the literature, it would seem reasonable to suggest that published contributions of peptides to the ruminal soluble N pool might have been overestimated because of incomplete precipitation or partial hydrolysis of soluble proteins by acid treatments.

In the present study, small peptides isolated by ultrafiltration contributed between 13 and 20% of the total AA in the soluble fraction and less than 3% of the total AA flowing at the omasal canal of cows fed diets differing in the concentration and source of supplemental protein. On the other hand, AA in soluble 3–10K oligopeptides averaged 71%, whereas FAA accounted for less than 2% of total soluble AA flows across diets. Therefore, our results suggest that hydrolysis of oligopeptides (3–10K) was the rate-limiting step during microbial degradation of soluble proteins and that small peptides and FAA were rapidly utilized by ruminal microbes and did not accumulate in the rumen. On average, 73% of total soluble AA and 10% of total AA flows were of dietary origin, indicating a substantial escape of dietary soluble AA from ruminal degradation and calling into question the use of in situ estimations of protein degradation to predict the flow of RUP. Although the omasal flow of some individual AA associated with peptides and oligopeptides was significantly affected by dietary treatment, these changes were relatively small compared with the total flow of AA in their soluble and insoluble forms. The methodology described in the present paper may allow for more accurate quantitation of the ruminal peptide and FAA pools, their variations caused by the diet, and their metabolic and nutritional significance to the ruminal microbes and the host animal.

	FOOTNOTES

 
¹ Mention of any trademark or proprietary product in this paper does not constitute a guarantee or warranty of the product by the USDA or the Agricultural Research Service and does not imply its approval to the exclusion of other products that may also be suitable.

³ Current address: Agricultural Research Service, USDA, US Dairy Forage Research Center, 1925 Linden Dr., Madison, WI 53706.

⁴ Current address: Lucta S. A., P. O. Box 1112, Barcelona 08080, Spain.

⁵ Current address: Granja Tres Arroyos, Buenos Aires, Argentina.

⁶ Current address: Dairy and Swine Research and Development Center, Agriculture and Agri-Food Canada, Lennoxville, Quebec, Canada J1M 1Z3.

Received for publication March 20, 2006. Accepted for publication September 20, 2006.

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