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Effects of Feeding Formate-Treated Alfalfa Silage or Red Clover Silage on Omasal Nutrient Flow and Microbial Protein Synthesis in Lactating Dairy Cows¹

A. F. Brito^*,2, G. A. Broderick^,3, J. J. Olmos Colmenero^*,4 and S. M. Reynal^*

^* Department of Dairy Science, University of Wisconsin, Madison 53706
Agricultural Research Service, USDA US Dairy Forage Research Center, 1925 Linden Drive West, Madison, WI 53706

³ Corresponding author: gbroderi{at}wisc.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Eight ruminally cannulated Holstein cows that were part of a larger lactation trial were blocked by days in milk and randomly assigned to replicated 4 x 4 Latin squares to quantify effects of nonprotein N (NPN) content of alfalfa silage (AS) and red clover silage (RCS) on omasal nutrient flows. Diets, fed as total mixed rations, contained 50% dry matter from control AS (CAS), ammonium tetraformate-treated AS (TAS), late maturity RCS (RCS1), or early maturity RCS (RCS2). Silages differed in NPN and acid detergent insoluble N (% of total N): 50 and 4% (CAS); 45 and 3% (TAS); 27 and 8% (RCS1); 29 and 4% (RCS2). The CAS, TAS, and RCS2 diets had 36% high-moisture shelled corn and 3% soybean meal, and the RCS1 diet had 31% high-moisture shelled corn and 9% soybean meal. All diets contained 10% corn silage, 27% neutral detergent fiber, and 17 to 18% crude protein. Compared with RCS, feeding AS increased the supply of rumen-degraded protein and omasal flows of nonammonia N and microbial protein, which may explain the improved milk yield observed in the companion lactation trial. However, omasal flow of rumen-undegraded protein was 34% greater on RCS. Except for Arg, omasal flows of individual AA, branched-chain AA, nonessential AA, essential AA, and total AA did not differ between cows fed AS vs. RCS. Within AS diets, no differences in omasal AA flows were observed. However, omasal flows of Asp, Ser, Glu, Cys, Val, Ile, Tyr, Lys, total nonessential AA, and total AA all were higher in cows fed RCS1 vs. cows fed RCS2. In this trial, there was no advantage to reducing NPN content of hay-crop silage.

Key Words: silage nonprotein nitrogen • omasal flow • microbial protein synthesis • dairy cow

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Greater concern regarding environmental N pollution has increased the importance of implementing nutritional strategies to minimize urinary N excretion. There is strong evidence that the high proportion of NPN in hay-crop silages results in depressed formation of microbial protein in the rumen when these are fed as the principal dietary forages (Dewhurst et al., 2000). Protein in alfalfa silage (AS) is extensively degraded to NPN (Muck, 1987). Nagel and Broderick (1992) showed that formic acid treatment of wilted AS decreased NPN formation and substantially improved N utilization when fed to lactating dairy cows. As a result of the polyphenol oxidase system in red clover (Jones et al., 1995), NPN formation is much reduced in red clover silage (RCS; Papadopoulos and McKersie, 1983; Albrecht and Muck, 1991). When RCS replaced dietary AS in 5 previous trials, energy digestibility was greater and there was evidence of improved N efficiency, although yield of milk and milk components was reduced in 2 trials and DMI was depressed in all 5 trials (Broderick et al., 2000, 2001).

We conducted 2 production studies (Broderick et al., 2007) to test the effects of using ammonium tetraformate (ATF) to reduce NPN content in AS and to compare AS (with about 50% of CP present as NPN) to RCS (with about 30% of CP present as NPN). The omasal sampling technique was used in the second of these trials with several objectives: 1) to determine the effects of feeding AS or RCS with differing NPN contents on ruminal digestibility and omasal flow of nutrients and microbial protein in lactating dairy cows; 2) to relate any differences in production and urinary N excretion in the parallel trial (Broderick et al., 2007) to differences in microbial protein synthesis and nutrient digesta flow; and 3) to compare NRC (2001) model predictions of RDP, RUP, and microbial NAN flow with in vivo measurements.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Animals and Diets
Eight ruminally cannulated multiparous Holstein cows, averaging (mean ± SD) 210 ± 37 DIM and 575 ± 44 kg of BW at the beginning of the study were blocked by DIM and randomly assigned to treatments within 2 replicated 4 x 4 Latin squares. These animals were part of a larger trial investigating effects of replacing dietary AS with RCS on milk production (Broderick et al., 2007). Each experimental period lasted 28 d, consisting of 14 d for diet adaptation and 14 d for sample collection, except for period 3, where 14 d were allotted for diet adaptation and 7 d for sample collection. This was necessary due to a shortage of one RCS and only 3 treatments were fed in the last period. Dietary sequences within Latin squares were organized to balance carryover effects in succeeding periods (i.e., each treatment followed every other treatment once in each square). Diets were fed as 4 TMR containing (% DM): 50.4% control AS (CAS), 9.9% rolled corn silage (CS), 36.1% rolled high-moisture shelled corn (HMSC), 3.2% solvent soybean meal (SSBM), and 17.7% CP; 50.4% ATF-treated AS (TAS), 9.9% CS, 36.1% HMSC, 3.2% SSBM, and 18.4% CP; 49.7% late maturity RCS (RCS1), 10% CS, 30.6% HMSC, 9.2% SSBM, and 17.2% CP; and 50.6% early maturity RCS (RCS2), 9.8% CS, 35.8% HMSC, 3.4% SSBM, and 17.2% CP. Diets averaged 27% NDF. Other details of the trial, including dietary ingredients and composition, are described in the companion report (Broderick et al., 2007). Care and handling of the animals including ruminal cannulation was conducted as outlined in the guidelines of the University of Wisconsin institutional animal care and use committee.

Sampling and Laboratory Analysis
Spot samples of omasal digesta leaving the rumen were obtained from the ruminally cannulated cows using the omasal sampling technique described by Huhtanen et al. (1997), Ahvenjärvi et al. (2000), and Reynal and Broderick (2005). The omasal digesta markers used were indigestible NDF (INDF; Huhtanen et al., 1994) for the large particle phase (LP), YbCl₃ (modified from Siddons et al., 1985) for the small particle phase (SP), and CoEDTA (Udén et al., 1980) for the fluid phase (FP). A marker solution containing YbCl₃, CoEDTA, and ¹⁵NH₄SO₄ with 95 atom % excess (APE) ¹⁵N (Isonics Corporation, Columbia, MD) was prepared as described by Reynal and Broderick (2005). Just before beginning marker infusion, samples of whole ruminal contents were taken from each cow to determine background abundance of ¹⁵N. The marker solution was continuously infused into the rumen for approximately 135 h at a constant rate of 2.66 L/d (providing 2.19 g of Co, 2.76 g of Yb, and 0.21 g of ¹⁵N/d) from d 21 to 26 (d 14 to 19 in period 3) using 2 syringe pumps (model no. 33; Harvard Apparatus, Inc., Holliston, MA). Omasal sampling began approximately 64 h after beginning marker infusion. Samples were taken 4 times daily at 2-h intervals over 3 consecutive days to represent the 24 h-feeding cycle: 0000, 0200, 0400, and 0600 h (d 24 in periods 1, 2, and 4; d 17 in period 3), 0800, 1000, 1200, and 1400 h (d 25 in periods 1, 2, and 4; d 18 in period 3), and 1600, 1800, 2000, and 2200 h (d 26 in periods 1, 2, and 4; d 19 in period 3). Before each sampling, it was necessary to confirm the location of the sampling tube and occasionally it had to be repositioned into the omasal canal. Sometimes it also was necessary to unplug the holes in the end of the sampling tube because of the presence of coarse digesta. At each sampling time, 200 mL of omasal digesta was collected, transferred to 3-L containers, and stored at –20°C. Spot samples were pooled over the 3 d to obtain a 2.4-L composite from each cow in each period for later separation into the omasal phases (LP, SP, and FP). At the second and fourth time points each day, 500-mL samples of omasal digesta were taken for isolation of bacteria and protozoa.

Protozoa were isolated using a modification of the procedure of Hristov et al. (2001). The first of the 500-mL samples was squeezed through 2 layers of cheesecloth and retained solids were washed with 500 mL of McDougall’s (1948) buffer, containing 0.5 g of glucose and 50 mg of cysteine-HCl/100 mL, that had been warmed to 39°C. Filtrates were transferred to 1-L separatory funnels and placed in a waterbath at 39°C. After approximately 45 min, separatory funnels were removed from the waterbath and the distinct white layers of sedimented protozoa forming at the bottom of each separatory funnel were carefully drawn off into vials and stored on ice until transported to the laboratory. At the laboratory, 20 mL of sucrose solution (30% wt/ vol) was added to 50-mL centrifuge tubes. Protozoal sediments were poured on top of the sucrose solution and tubes were centrifuged at 150 x g for 3 min at 4°C. Supernatants were discarded and the pellets washed 3 times with saline (0.85% wt/vol of NaCl) followed by centrifugation at 1,200 x g for 5 min at 4°C. These pellets were frozen for about 12 h, then transferred to vials and stored at –20°C until freeze-dried. Fluid-associated bacteria (FAB) and particle-associated bacteria (PAB) were isolated from the second of the daily 500-mL samples obtained from each cow on each sampling day using filtration and differential centrifugation as described earlier in detail (Reynal and Broderick, 2005). Both the FAB and PAB pellets were stored at –20°C until freeze-drying. After freeze-drying, bacterial and protozoal samples were ground with a mortar and pestle and equal DM from each was pooled for each cow in each period for later analysis.

The 2.4-L pooled omasal composites were thawed at room temperature and separated into the 3 omasal phases (LP, SP, and FP) as described by Reynal et al. (2003), and were stored at –20°C until freeze-dried. After freeze-drying, these samples were ground through a 1-mm screen (Wiley mill; Arthur H. Thomas, Philadelphia, PA) and analyzed for markers. Concentrations of Co, Yb, and INDF in LP and SP, and Co and Yb in FP were determined using the methods detailed earlier (Reynal and Broderick, 2005). Marker concentrations were used to physically recombine DM from the freeze-dried FP, SP, and LP in the correct proportions to reconstitute the omasal true digesta (OTD) flowing out of the rumen using the triple-marker method of France and Siddons (1986). Concentrations of Co, Yb, and INDF were distinctly greater in, respectively, the FP, SP, and LP, thus allowing for successful application of the triple-marker method.

Omasal true digesta samples were analyzed for total N (Leco 2000, Leco Instruments, Inc., St. Joseph, MI), absolute DM (105°C), ash, and OM (AOAC, 1980), sequentially for NDF and ADF using heat stable -amylase and Na₂SO₃ (Hintz et al., 1995), and for neutral detergent insoluble nitrogen (NDIN) and ADIN. Extracts also were prepared from OTD samples as follows: 10 mL of pH 2.2 buffer (77.5 mM Na citrate) was added to 0.5 g of dry sample and then vortexed. After 30 min at 39°C, extracts were centrifuged (15,000 x g, 15 min, 4°C), and supernatants stored at –20°C for later analysis of ammonia and total free AA using assays (Broderick and Kang, 1980) adapted to flow-injection analysis (Lachat Quik-Chem 8000 FIA; Lachat Instruments, Milwaukee, WI). For determination of individual AA, OTD samples were hydrolyzed for 24 h at 110°C in vials containing 6 N HCl with 0.1% (wt/vol) phenol (Mason et al., 1979) sealed under a N₂ atmosphere. The ratio of sample to acid was about 1 mg of total N per 5 mL of 6 N HCl. After hydrolysis, samples were cooled, HCl removed by evaporation and the residue redissolved in pH 2.2-sample buffer containing norleucine as internal standard. Analysis of individual AA was conducted using ion exchange chromatography with ninhydrin detection (Beckman 6300 Amino Acid Analyzer; Beckman Instruments, Inc., Palo Alto, CA).

Samples of FAB, PAB, protozoa, and OTD were prepared for total NAN and ¹⁵N analyses as follows: approximately 100 µg of N from each sample was weighed into tin cups (Elemental Microanalysis Ltd., Okehampton, UK) followed by addition of 50 µL of 72 mM K₂CO₃. The tin capsules were placed in 96-well microtiter plates and dried in a 60°C oven overnight to volatilize ammonia. Samples of ruminal digesta collected before infusion for determination of background abundance of ¹⁵N were freeze-dried and ground to pass both a 1-mm Wiley mill screen and then a 0.5-mm Udy mill screen (Udy cyclone mill; Udy Corp., Fort Collins, CO). These samples also were prepared for NAN and ¹⁵N analysis as described above. Nitrogen and ¹⁵N were determined using a Carlo-Erba instrument interfaced to an isotope ratio mass spectrometer (University of California-Davis Stable Isotope Facility). Bacteria (FAB and PAB), protozoa, FP, and OTD also were analyzed for absolute DM, ash, and OM (AOAC, 1980).

Calculations
Omasal flow of NAN was determined by difference between total N and ammonia N flows. Total NAN flow at the omasum was assumed to be composed of PAB NAN, FAB NAN, and nonammonia nonmicrobial N (NANMN). The mean (±SD) background ¹⁵N during trial was 0.36797 (±0.000259) atom %. Thus, the ¹⁵N APE above background (¹⁵N enrichment) for samples from each cow in each period was calculated:

The FAB and PAB were assumed to be representative of bacteria flowing with, respectively, the FP and the SP plus LP (SP + LP). The particulate phase (PF) fractions used to compute PAB NAN flowing with the SP + LP were obtained by mixing pellets from the 1,000 x g centrifugation step from FAB isolation (Reynal and Broderick, 2005) with the residues retained when the daily 500-mL omasal samples (used in bacterial isolation) were filtered through single layers of 55-µm Dacron mesh. The 1,000 x g pellets were assumed to represent the small particles, and the residues retained on 55-µm Dacron mesh were assumed to represent the large particles. The PF fractions were also freeze-dried, sequentially ground through 1- and 0.5-mm screens, and analyzed for DM, ash, OM, NAN, and ¹⁵N as described before. Omasal flows of PAB NAN and FAB NAN were calculated as:

where FP APE was computed using the equations:

This was done because centrifugation at 10,000 x g was used to separate FP from SP when the 3 phases were isolated from pooled 2.4-L omasal samples (Reynal et al., 2003). Total microbial NAN, NANMN and RUP flows at the omasal canal, RDP supply, OM truly digested in the rumen (OMTDR), and efficiency of microbial NAN synthesis were calculated as follows:

where flows and intakes are in grams per day or kilograms per day and NAN concentrations are in grams per gram of OM.

Statistical Analysis
Data were analyzed using Proc Mixed in SAS (SAS Institute, 1999–2000) for a replicated 4 x 4 Latin square design according to the following model:

where Y_ijkl = dependent variable, µ = overall mean, S_i = effect of square i, P_j = effect of period j, C_k(i) = effect of cow k (within square i), T_l = effect of treatment l, ST_il = interaction between square i and treatment l, and E_ijkl = residual error. All terms were considered fixed, except C_k(i) and E_ijkl, which were considered random. The inter-action term was removed from the model when P 0.25. Preplanned, single degree of freedom orthogonal contrasts were constructed to assess effects of ATF treatment of AS (CAS vs. TAS; ATF contrast), RCS maturity (RCS1 vs. RCS2; maturity contrast), and silage source (CAS + TAS vs. RCS1 + RCS2; silage contrast). Significance was declared at P 0.05 and trends at 0.05 < P 0.10. All reported values are least squares means. When standard errors of the differences of the least squares means were not equal due to unequal replication, the largest standard error was reported.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Omasal Nutrient Flow and Ruminal Digestibility
No differences were observed for DMI, DM flow, and DM apparently digested in the rumen, which averaged, respectively, 21.4, 13.5, and 8.0 kg/d across diets (Table 1). However, a trend (P = 0.10) for a greater amount of DM apparently digested in the rumen was detected in cows fed RCS2 vs. RCS1, possibly because of its earlier maturity. Greater DMI was observed in cows fed AS in the parallel production trial conducted with 24 animals (Broderick et al., 2007); lack of significance for DMI was probably due to fewer animals (8 ruminally cannulated cows) being used in this study. When ruminal DM digestibility was expressed as a percentage of DMI, there was a trend (P = 0.09) for greater digestibility on CAS vs. TAS, whereas digestibility on RCS1 was lower (P = 0.02) than that on RCS2. No significant silage effect was observed; however, apparent total tract digestibility of DM was higher on RCS than AS (P < 0.01; Broderick et al., 2007), suggesting greater intestinal DM digestion on the RCS diets. It is important to note that INDF was used as the internal marker for assessing ruminal digestibility whereas indigestible ADF was used to estimate total tract digestibility. Beside the different internal markers, pore size of the indigestible ADF bags was larger (21 µm) than for INDF bags (6 µm). Therefore, differences in marker methodology may account for some of these results. Dewhurst et al. (2003) reported that cows fed diets based on RCS or AS had apparent ruminal DM digestibilities of, respectively, 36 and 28%. These authors also showed that ruminal outflow rate was more rapid on AS (6.1%/h) compared with RCS (4.8%/h), which might explain the lower ruminal DM digestibility observed on AS in their trial.

Although NDF intake was similar among diets, cows fed AS consumed, on average, 0.48 kg/d more ADF (P = 0.01; Table 1) than those fed RCS, partly reflecting the greater ADF content of AS diets (Table 2; Broderick et al., 2007). Omasal flows of both NDF and ADF were higher (P < 0.01) on AS than on RCS diets, whereas the opposite was true for ruminal fiber digestibility (Table 1). Dewhurst et al. (2003) showed that a greater proportion of particles <2 mm were present in the rumen of cows fed the AS diet compared with the RCS diet and more small particles might have contributed to the more rapid passage rate observed for AS. This might have accounted for the lower fiber digestibility on AS in the present study. Dewhurst et al. (2003) speculated that thicker ruminal mats on RCS vs. AS resulted in greater entrapment of feed particles, longer ruminal retention times, lower passage rates, and greater fiber digestibility. Presumably, cows derive more energy with greater fiber digestion and, thus, should produce more milk. However, cows yielded less milk and milk components on RCS (Broderick et al., 2007), despite greater ruminal and total tract digestibility of fiber on RCS diets. This is somewhat difficult to explain but may be related to differences in energy partitioning between the 2 silage sources. It can be speculated that energy derived from this greater fiber digestibility was channeled to fat deposition rather than production, which is supported by the greater BW gains (P < 0.01; Broderick et al., 2007) in cows fed RCS instead of AS. As was observed for DM and OM, apparent ruminal digestibility of NDF and ADF as a proportion of intake also was lower on RCS1 than on the less mature RCS2 (Table 1).

Flow of FAB NAN was higher (P = 0.04) on RCS diets than on AS diets, whereas the opposite was true for PAB NAN flow (P < 0.01; Table 2). The FAB NAN averaged 29 (AS diets) and 41% (RCS diets) and PAB NAN 71% (AS diets) and 59% (RCS diets) of the total microbial NAN flow, which differed significantly between silage pairs (Table 2). Reynal and Broderick (2005) also observed that total microbial NAN flow was contributed mainly from PAB. Craig et al. (1987), using ¹⁵N as the microbial marker to distinguish between FAB and PAB, observed that 70 to 80% of microbial OM in whole ruminal contents was associated with the particulate phase. However, Hristov and Broderick (1996) found similar contributions from PAB and FAB to the total microbial NAN flow, possibly because the outflow rate of the fluid phase was 3 times greater than that of the particulate phase, which may also explain the greater contribution from FAB than PAB in the studies of Ahvenjärvi et al. (2002) and Olmos Colmenero and Broderick (2006). Discrepant results can also be associated with digesta flow measurements, methodology used to isolate bacteria, bacterial marker, and diet composition.

Omasal flow of total microbial NAN was, on average, 69 g/d greater (P < 0.01) in cows fed AS compared with those fed RCS, and 78 and 66% of the total NAN flow was contributed by microbial NAN on AS and RCS diets (P < 0.01), respectively (Table 2). However, no difference was observed in total microbial NAN flow within AS and RCS. However, Dewhurst et al. (2003) found that cows fed diets containing RCS (either RCS alone or 50:50 mixtures of RCS plus white clover) yielded numerically greater microbial NAN flows than cows fed AS diets. Greater microbial protein synthesis on AS in the present trial may have been due to greater RDP supply on CAS and TAS diets (Table 2). Supply of RDP averaged 3,188 and 2,487 g/d, respectively, for AS and RCS diets (P < 0.01). Santos et al. (1998) reported that, in 29 comparisons from 15 metabolism trials, microbial N flow to the duodenum was significantly decreased by feeding high RUP sources in 10 comparisons and was numerically higher for soybean meal in 25 comparisons, indicating that replacing soybean meal with supplements rich in RUP might limit microbial protein synthesis due inadequate RDP supply. Ipharraguerre and Clark (2005), using a meta-analysis approach, found an overall 7% depression (P < 0.05) in microbial NAN flow to the small intestine in response to RUP supplementation of dairy cows. Reynal and Broderick (2005) showed that reducing the RDP supply from 3,076 to 2,403 g/d, by replacing SSBM with expeller soybean meal, decreased the omasal flow of microbial NAN from 470 to 384 g/d. Olmos Colmenero and Broderick (2006) observed a linear increase in microbial NAN flow from 425 to 480 g/d when dietary RDP increased from 1,979 to 3,028 g/d.

Reduced RDP supply on RCS may have depressed microbial protein synthesis via lower ruminal concentrations of ammonia and total free AA on those diets (Broderick et al., 2007). Satter and Slyter (1974) reported that mixed populations of ruminal bacteria reach maximum yields of cell protein if the ruminal fluid has a minimum concentration of 5 mg of ammonia N/dL. Kang-Meznarich and Broderick (1980) found that microbial protein synthesis in the rumen was maximal at 8.5 mg ammonia N/dL. Ruminal ammonia N on both AS diets was never below 5 mg/dL and was greater than 8.5 mg/dL at all times except at prefeeding and 24 h postfeeding on CAS, and at prefeeding, 18, and 24 h postfeeding on TAS (Figure 1). However, ruminal ammonia N was lower than 8.5 mg/dL at prefeeding and from 4 to 24 h postfeeding on RCS1 and at prefeeding and from 8 to 24 h postfeeding on RCS2 (Figure 1). Ammonia is often the main N source for microbial protein synthesis (Nolan, 1975; Aharoni et al., 1991) and is essential for the growth of several species of ruminal bacteria (Blackburn, 1965; Allison, 1970; Bryant, 1973); however, preformed AA and peptides stimulated bacterial growth and yield in vivo (Chikunya et al., 1996) and in vitro (Argyle and Baldwin, 1989; Carro and Miller, 1999). In the present trial, greater (P < 0.01) omasal flow of total free AA N was observed on AS diets, which averaged 23 g/d vs. 15 g/d on the RCS diets (Table 2). Greater ruminal concentrations of total free AA N also were found on AS vs. RCS diets (Broderick et al., 2007). Thus, these data strongly suggest that increased RDP supply contributed more ruminal ammonia and free AA, and possibly peptides, for microbial protein synthesis.

View larger version (27K): [in this window] [in a new window]   Figure 1. Effects of dietary forage source on ruminal ammonia concentrations after feeding (refer to Broderick et al., 2007, for further details). CAS = control alfalfa silage; TAS = ammonium tetraformate-treated alfalfa silage; RCS1 = red clover silage 1 (late maturity); RCS2 = red clover silage 2 (early maturity). Error bars = ± 1 SEM. A significant time x diet interaction (P < 0.01) was observed. Significant differences among treatments, which are indicated in the figure (*), occurred at: 1 h and 2 h (CAS and TAS were greatest, RCS2 was intermediate, and RCS1 was lowest); at 4 h (CAS was greatest, TAS was similar to CAS and RCS2 but different from RCS1, and RCS2 was different from RCS1); and at 8, 12, and 18 h (CAS and TAS were greater and RCS1 and RCS2 were lower).

Microbial N synthesis estimated using the NRC (2001) model averaged 327 and 308 g/d, respectively, for the AS and RCS diets, a mean overall underprediction of 18% compared with microbial yields measured using ¹⁵N (Table 3). Although the NRC (2001) model predicted a reduction of only 19 g/d of microbial NAN flow on RCS, the actual decrease observed was 69 g/d. Reynal et al. (2003) also reported that the NRC (2001) model underpredicted microbial protein synthesis by 50 to 89% when total purines were used as the internal microbial marker. Similarly, Reynal and Broderick (2005) found that microbial N flows predicted by the NRC (2001) model were lower than in vivo measurements using ¹⁵N as the bacterial marker. These apparently low estimates of microbial protein synthesis by the NRC (2001) model might be related to the bacterial marker used in its measurement. Most of the data used to develop the NRC (2001) prediction for microbial protein synthesis derived from studies in which microbial protein was quantified with the Zinn and Owens (1986) purine assay. Obispo and Dehority (1999) and Makkar and Becker (1999) both reported low recoveries of total purines using the Zinn and Owens (1986) method. Disproportionate recoveries of purines between bacterial and duodenal samples could lead to underestimates of microbial flow according to Reynal et al. (2003) who modified the Zinn and Owens (1986) assay to improve purine recovery in omasal samples. In addition, Reynal et al. (2005) reported less reliable estimates of microbial protein flow when total purines rather than ¹⁵N were used as marker. Furthermore, many factors influencing microbial growth in the rumen, such as source and amount of RDP, ruminal pH and dilution rate, source and amount of dietary carbohydrate, and excessive levels of dietary fat, are not considered by the NRC (2001) model, which may lead to discrepancies between predicted and observed microbial protein flows.

The NRC (2001) model predicted a mean RDP supply that was 8.3% higher than that measured in vivo across all diets (Table 3); however, RDP supply was underpredicted by only 1.1% for CAS but overpredicted by 9.4, 16, and 10% for, respectively, TAS, RCS1, and RCS2. The NRC (2001) model overpredicted RUP flow by 36 and 23% for CAS and TAS and underpredicted RUP flow by 6.0 and 7.2% for RCS1 and RCS2 (Table 3). In the present trial, RUP flow was calculated by subtracting omasal microbial NAN flow from total N flow. Therefore, any inaccuracy in determination of microbial NAN flow would result in inaccurate RUP measurement and could have contributed to these differences. Moreover, RUP flows in the NRC (2001) model were computed from in situ data. Because of differences in methodologies, discrepancies between RUP flow estimated in situ vs. omasal RUP flow measured in vivo are to be expected and comparisons should be made cautiously.

AA Flow
Except for Arg, omasal flow of individual AA as well as omasal flow of branched-chain AA (BCAA), nonessential AA (NEAA), essential AA (EAA), and total AA did not differ significantly between silage pairs (Table 4). Omasal flow of Glu (P = 0.10) and His (P = 0.08) tended to be greater on cows fed RCS compared with AS diets. Based on these findings, the greater yields of 1.5 kg/d more milk and 95 g/d more protein observed when AS replaced RCS (Broderick et al., 2007) cannot be explained by differences in AA supply. The AA determined in the OTD were contributed by microbial protein, feed protein escaping ruminal degradation, and, to a lesser extent, endogenous protein. The lack of a significant silage effect on omasal AA flows probably occurred because greater NANMN supply (feed RUP plus endogenous protein) on RCS was counterbalanced by greater microbial NAN supply on AS (Table 2). Replacing dietary soybean meal with RUP increased the intestinal supply of NANMN but decreased that of microbial NAN (Ipharraguerre and Clark, 2005). Differences in yields of milk and milk protein observed in the companion lactation trial (Broderick et al., 2007) might have occurred because the profile of absorbed AA was closer to the EAA requirements of lactating cows (NRC, 2001) on AS diets. Microbial protein has an AA profile more closely resembling that of milk protein (Santos et al., 1998) and microbial NAN flow was greater when AS replaced RCS in the diet (Table 2). According to Clark et al. (1992), the CP source modulates the intestinal AA supply by altering flows of RUP and microbial N to the lower gastrointestinal tract. In addition, the contribution of RUP to total protein passage, and RUP AA composition, influence the pattern of AA absorbed at the small intestine (Rulquin and Verite, 1993; NRC, 2001). Therefore, it is also possible that the AA profile of RUP in cows fed AS was more complementary to microbial protein, thus improving milk production. However, differences in milk yield between silages sources may also be explained by greater energy intake: cows fed CAS and TAS ingested on average 1.4 kg/d more DM and 2.1 Mcal/d more NE_L than those fed RCS1 and RCS2 (Broderick et al., 2007).

Duodenal flows of Lys and Met predicted by the NRC (2001) model averaged 188 and 54 g/d among diets indicating an overall difference of 29 and 12% between in vivo and predicted data, respectively (Table 3). According to the NRC (2001) model, the optimal use of MP for both maintenance and milk protein production requires concentrations of Lys and Met in MP that approximate 7.2 and 2.4%, respectively, for an optimum MP Lys:Met ratio of 3:1. The measured Lys:Met ratio averaged 2.8:1, whereas that predicted by the NRC (2001) model was 3.5:1 across diets (Table 3), suggesting that Lys may have been marginally limiting on this trial. However, it should be noted that the observed Lys:Met ratios were closer to the optimum of 3:1 than those predicted by the NRC (2001) model. Although the Lys:Met ratio tended (P = 0.08; Table 4) to be greater and was closer to the optimum on RCS, lower production on those diets also suggested that some Lys may have been unavailable for synthesis of milk and milk protein at the mammary gland.

Microbial Composition
No differences due to forage source were observed for the OM content of FAB, which averaged 82% across diets (Table 5). However, OM content of PAB was significantly higher (P = 0.01) for AS than RCS diets. In addition, a trend (P = 0.08) for greater OM content in PAB was found for RCS2 compared with RCS1. Organic matter content of PAB was numerically higher than that of FAB, which was in agreement with previous reports (Martin et al., 1994; Rodriguez et al., 2000). The NAN content of FAB was greater (P < 0.01) in cows fed AS rather than RCS (Table 5). Conversely, feeding either AS or RCS diets did not affect the NAN content of PAB. Within silages, RCS1 gave rise to greater (P < 0.01) PAB NAN content than RCS2, whereas no difference was found between CAS and TAS. The ¹⁵N enrichment of both FAB and PAB were lower (P < 0.01) on AS than on RCS diets, which may reflect greater dilution from the larger ammonia N pool when AS was fed (Broderick et al., 2007). On average, ¹⁵N enrichment of FAB was 18% higher than PAB (Table 5). This numerical difference could be attributed to differential utilization by PAB and FAB of free ammonia N in the FP. Microbial species that are associated primarily with particulate matter may also utilize greater amounts of AA and peptides as N sources, thus diluting the ¹⁵N-ammonia. Other workers (Hristov and Broderick, 1996; Ahvenjärvi et al., 2002; Reynal and Broderick, 2005) have also observed greater ¹⁵N enrichment of FAB; therefore, differential isotopic enrichments of FAB and PAB must be accounted for when estimating microbial NAN flows from the rumen.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Replacing dietary AS with RCS resulted in greater ruminal fiber digestibility as well as reduced RDP and increased RUP supplies. Reducing NPN content of AS by ATF treatment did not appear to alter dietary RDP or RUP supply. Microbial NAN flow was greater on AS, suggesting that reduced NPN or increased RUP contents of RCS may have depressed microbial protein synthesis in the rumen. Generally, there were no differences in omasal AA flow when AS and RCS were fed, suggesting that greater RUP flow was compensated for by reduced microbial protein formation in cows fed RCS. Greater omasal flow of RUP and most AA on the more mature RCS may have resulted from its greater content of NDIN, ADIN, and fraction B₃. Increased yield of milk and milk protein on AS (Broderick et al., 2007) may have been the result of greater supply of nutritionally available AA from high quality microbial protein. The NRC (2001) model and urinary PD excretion underestimated microbial protein synthesis by 18 and 32%, respectively, compared with that measured in vivo using ¹⁵N. Moreover, significant differences in microbial protein synthesis between AS and RCS were not detected using PD excretion.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The authors thank Rick Walgenbach and the farm crew for harvesting and storing the feeds and Len Strozinski for animal care and sampling at the US Dairy Forage Research Farm (Prairie du Sac, WI); Wendy Radloff, Fern Kanitz, Mary Becker, and Adam Ford for help in the samplings and laboratory analysis; and Peter Crump for assisting with statistical analysis.

	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 also may be suitable.

² Current address: Agriculture and Agri-Food Canada, PO Box 90, 2000 Route 108 Est, Lennoxville, QC-Canada.

⁴ Current address: Centro Universitario de los Altos, Universidad de Guadalajara, Tepatitlan de Morelos, Jalisco, Mexico CP 47600.

Received for publication June 2, 2006. Accepted for publication September 15, 2006.

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