TY - JOUR
AU - Grandeen, K. L.
AB - ABSTRACT The objectives of this experiment were to investigate the effects of two ruminally degradable protein (RDP) levels in diets containing similar ruminally undegradable protein (RUP) and metabolizable protein (MP) concentrations on ruminal fermentation, digestibility, and transfer of ruminal ammonia N into milk protein in dairy cows. Four ruminally and duodenally cannulated Holstein cows were allocated to two dietary treatments in a crossover design. The diets (adequate RDP [ARDP] and high RDP [HRDP]), had similar concentrations of RUP and MP, but differed in CP/RDP content. Ruminal ammonia was labeled with 15N and secretion of tracer in milk protein was determined for a period of 120 h. Ammonia concentration in the rumen tended to be greater (P = 0.06) with HRDP than with ARDP. Microbial N flow to the duodenum, ruminal digestibility of dietary nutrients, DMI, milk yield, fat content, and protein content and yield were not statistically different between diets. There was a tendency (P = 0.07) for increased urinary N excretion, and blood plasma and milk urea N concentrations were greater (P = 0.002 and P = 0.01, respectively) with HRDP compared with ARDP. Milk N efficiency was decreased (P = 0.01) by the HRDP diet. The cumulative secretion of ammonia 15N into milk protein, as a proportion of 15N dosed intraruminally, was greater (P = 0.003) with ARDP than with HRDP. The proportions of bacterial protein originating from ammonia N and milk protein originating from bacterial or ammonia N averaged 43, 61, and 26% and were not affected by diet. This experiment indicated that excess RDP in the diet of lactating dairy cows could not be efficiently utilized for microbial protein synthesis and was largely lost through urinary N excretion. At a similar MP supply, increased CP or RDP concentration of the diet would result in decreased efficiency of conversion of dietary N into milk protein and less efficient use of ruminal ammonia N for milk protein syntheses. Introduction Increasing the CP content of dairy cow diets may result not only in greater milk production (Armentano et al., 1993; Wu and Satter, 2000), but also in increased concentrations of ruminal ammonia and blood urea N and consequently greater urinary N losses (Armentano et al., 1993; Christensen et al., 1993; Castillo et al., 2001). Although, as exemplified by Tamminga (1992), ruminal N loss is the greatest single contributor to urinary N losses, metabolic losses, indigestible microbial N, losses in maintenance, and inefficient conversion of absorbed AA into milk protein may comprise up to 72% of the urinary N losses in the dairy cow. With few exceptions, in studies investigating effects of CP level, diets supplied different amounts of metabolizable (MP), ruminally degradable (RDP), or ruminally undegradable protein (RUP). Thus, the individual contributions of RDP, RUP, or MP to urinary or overall N losses cannot be readily distinguished. Dietary RDP can be used for microbial protein synthesis (MPS), provided energy is not limiting. If not used for MPS, RDP can be converted to ammonia, absorbed through the ruminal wall, detoxified to urea in the liver (Lobley et al., 1995) and largely lost in urine; some RDP may bypass the rumen and contribute to the duodenal AA and peptide flow (Choi et al., 2002). Therefore, the efficiency of RDP use in the rumen is a central factor determining the economic cost and environmental impact of ruminant production. We hypothesized that, provided energy is not limiting in the rumen, excess ammonia from feed RDP will enhance MPS and its use for milk protein synthesis by the dairy cow. Thus, the objectives of this study were to investigate the effect of dietary RDP level in diets with similar and presumably adequate RUP and MP concentrations on ruminal fermentation, microbial protein outflow from the rumen, nutrient digestibility, and transfer of ruminal ammonia N into milk protein by lactating dairy cows. Materials and Methods Animals and Feeding Four multiparous, late-lactation Holstein cows fitted with 10-cm ruminal (Bar Diamond, Parma, ID) and simple T-type duodenal (Ankom Technology, Fairport, NY) cannulas were used in this experiment. The duodenal cannulas were placed on the ascending duodenum, anterior to the pancreatic duct. The cows (average ±SE; BW 757 ± 61 kg; DIM 257 ± 68 d), cared for according to the guidelines of the University of Idaho Animal Care and Use Committee, were grouped (two cows per group) and fed specific diets in a crossover design. Treatments were diets (Table 1) with adequate RDP (ARDP) or high RDP (HRDP). Diets had similar concentrations of RUP and estimated MP, but diet HRDP provided greater levels of CP and RDP than diet ARDP. Similar concentrations of RUP were achieved by including Soy-Pass (a soybean meal [SBM] treated with lignosulfonates, Borregaard Lignotech U.S.A., Rothschild, WI) in the ARDP diet. Cows were fed at 0600 and 1800 at 95% of ad libitum intake determined before initiation of each experimental period. Diets were mixed in a Data Ranger (American Calan Inc., Northwood, NH). Refusals, if any, were collected and weighed daily; composited weekly samples (per cow and per period) were analyzed for chemical composition. Each experimental period comprised 14 d for adaptation to the diet and 7 d for sampling. Table 1. Composition of the diets used in the experiment   Dieta  Item  ARDP  HRDP  Ingredient, % of DM      Alfalfa hay, choppedb  24.0  26.2      Triticale silageb  23.6  23.8      Corn grain, steam-rolled  30.1  25.0      Cottonseed, whole with lint  8.0  6.8      Solvent soybean meal, 48% CP  3.4  10.5      Nonenzymatically brown SBMc  3.0  0.0      Sugarcane molasses, dry  3.0  3.0      Dicalcium phosphate  0.5  0.5      Monosodium phosphate  0.2  0.2      Sodium bicarbonate  2.1  2.0      Salt  0.5  0.5      Mineral/vitamin premixd  1.6  1.5  Composition, % of DM      NEl, Mcal/kge  1.58  1.58      MPe  10.8  10.8      CP  15.8  18.3      RUPf  6.4  6.7      RDPf  9.4  11.6      NDF  34.2  34.5      ADF  23.9  26.8      Ash  8.3  8.3      Starch  18.0  16.7      Ether extract  3.9  3.7      Lignin  3.4  3.8      ANDFg  26.0  25.4      NFCh  37.8  35.2    Dieta  Item  ARDP  HRDP  Ingredient, % of DM      Alfalfa hay, choppedb  24.0  26.2      Triticale silageb  23.6  23.8      Corn grain, steam-rolled  30.1  25.0      Cottonseed, whole with lint  8.0  6.8      Solvent soybean meal, 48% CP  3.4  10.5      Nonenzymatically brown SBMc  3.0  0.0      Sugarcane molasses, dry  3.0  3.0      Dicalcium phosphate  0.5  0.5      Monosodium phosphate  0.2  0.2      Sodium bicarbonate  2.1  2.0      Salt  0.5  0.5      Mineral/vitamin premixd  1.6  1.5  Composition, % of DM      NEl, Mcal/kge  1.58  1.58      MPe  10.8  10.8      CP  15.8  18.3      RUPf  6.4  6.7      RDPf  9.4  11.6      NDF  34.2  34.5      ADF  23.9  26.8      Ash  8.3  8.3      Starch  18.0  16.7      Ether extract  3.9  3.7      Lignin  3.4  3.8      ANDFg  26.0  25.4      NFCh  37.8  35.2  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Triticale silage was (as-fed basis) 32.0% DM, 15.5% CP, and 59.2% NDF. Alfalfa hay was 84.0% DM, 20.6% CP, and 36.1% NDF. c SoyPass, Borregaard Lignotech USA, Rothschild, WI. d Composition: 34.5 to 41.4% NaCl; 26.9 to 33.8% ground rice hulls; 27.9% barley; 0.24% MnSO4; 0.21% ZnO3; 0.052% Cu2SO4; 0.0014% Fe2CO3; 0.0059% CaI2O6•H2O; 0.0010% CoCO3; 932,000 IU/kg of vitamin A; 174,000 IU/kg of vitamin D; and 3,300 U/kg of vitamin E. e MP = metabolizable protein; estimates for MP and NEl from NRC (2001). f Ruminally undegradable protein (RUP) estimated with an inhibitor in vitro system from net release of NH3 and total AA, assuming a ruminal passage rate of 0.06 h−1 (Broderick, 1987). Ruminally degradable protein calculated as 100 minus RUP. g ANDF (available NDF) calculated as NDF −(lignin × 2.4) (CPM Dairy v. 2.0.23; Univ. of Pennsylvania, Kennett Square, PA; Cornell Univ., Ithaca, NY; and William H. Miner Agric. Res. Inst., Chazy, NY). h NFC (nonfibrous carbohydrates) calculated as 100 − CP − NDF − ash − ether extract (NRC, 2001). View Large Table 1. Composition of the diets used in the experiment   Dieta  Item  ARDP  HRDP  Ingredient, % of DM      Alfalfa hay, choppedb  24.0  26.2      Triticale silageb  23.6  23.8      Corn grain, steam-rolled  30.1  25.0      Cottonseed, whole with lint  8.0  6.8      Solvent soybean meal, 48% CP  3.4  10.5      Nonenzymatically brown SBMc  3.0  0.0      Sugarcane molasses, dry  3.0  3.0      Dicalcium phosphate  0.5  0.5      Monosodium phosphate  0.2  0.2      Sodium bicarbonate  2.1  2.0      Salt  0.5  0.5      Mineral/vitamin premixd  1.6  1.5  Composition, % of DM      NEl, Mcal/kge  1.58  1.58      MPe  10.8  10.8      CP  15.8  18.3      RUPf  6.4  6.7      RDPf  9.4  11.6      NDF  34.2  34.5      ADF  23.9  26.8      Ash  8.3  8.3      Starch  18.0  16.7      Ether extract  3.9  3.7      Lignin  3.4  3.8      ANDFg  26.0  25.4      NFCh  37.8  35.2    Dieta  Item  ARDP  HRDP  Ingredient, % of DM      Alfalfa hay, choppedb  24.0  26.2      Triticale silageb  23.6  23.8      Corn grain, steam-rolled  30.1  25.0      Cottonseed, whole with lint  8.0  6.8      Solvent soybean meal, 48% CP  3.4  10.5      Nonenzymatically brown SBMc  3.0  0.0      Sugarcane molasses, dry  3.0  3.0      Dicalcium phosphate  0.5  0.5      Monosodium phosphate  0.2  0.2      Sodium bicarbonate  2.1  2.0      Salt  0.5  0.5      Mineral/vitamin premixd  1.6  1.5  Composition, % of DM      NEl, Mcal/kge  1.58  1.58      MPe  10.8  10.8      CP  15.8  18.3      RUPf  6.4  6.7      RDPf  9.4  11.6      NDF  34.2  34.5      ADF  23.9  26.8      Ash  8.3  8.3      Starch  18.0  16.7      Ether extract  3.9  3.7      Lignin  3.4  3.8      ANDFg  26.0  25.4      NFCh  37.8  35.2  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Triticale silage was (as-fed basis) 32.0% DM, 15.5% CP, and 59.2% NDF. Alfalfa hay was 84.0% DM, 20.6% CP, and 36.1% NDF. c SoyPass, Borregaard Lignotech USA, Rothschild, WI. d Composition: 34.5 to 41.4% NaCl; 26.9 to 33.8% ground rice hulls; 27.9% barley; 0.24% MnSO4; 0.21% ZnO3; 0.052% Cu2SO4; 0.0014% Fe2CO3; 0.0059% CaI2O6•H2O; 0.0010% CoCO3; 932,000 IU/kg of vitamin A; 174,000 IU/kg of vitamin D; and 3,300 U/kg of vitamin E. e MP = metabolizable protein; estimates for MP and NEl from NRC (2001). f Ruminally undegradable protein (RUP) estimated with an inhibitor in vitro system from net release of NH3 and total AA, assuming a ruminal passage rate of 0.06 h−1 (Broderick, 1987). Ruminally degradable protein calculated as 100 minus RUP. g ANDF (available NDF) calculated as NDF −(lignin × 2.4) (CPM Dairy v. 2.0.23; Univ. of Pennsylvania, Kennett Square, PA; Cornell Univ., Ithaca, NY; and William H. Miner Agric. Res. Inst., Chazy, NY). h NFC (nonfibrous carbohydrates) calculated as 100 − CP − NDF − ash − ether extract (NRC, 2001). View Large 15N-dosing, Sampling, and Related Analyses Ruminal ammonia N was labeled through a pulse dose of 10 g of 20 atom percent excess (15NH4)2SO4 (Cambridge Isotope Laboratories, Inc., Andover, MA) dissolved in 7 L of McDougall's buffer (McDougall, 1948). The rumen of each cow was evacuated in large-capacity cart on d 15 before the 0600 feeding; contents were weighed, a background ruminal sample was taken following a thorough mixing by hand, and the isotope was added to the ruminal contents that were returned to the rumen. Compared with our previous approach (continuous intraruminal infusion; Hristov and Ropp, 2003; Hristov et al., 2004a), this technique of dosing the isotope produced considerably less variability in 15N-enrichment of ruminal ammonia N (Figure 1) and gave us the ability to accurately estimate the area under the 15N-enrichment curve (AUC) for this N pool. Figure 1. View largeDownload slide Effect of ruminally degradable protein (RDP) on 15N-enrichment of ammonia N in dairy cows (means ±SE). • = adequate-RDP diet (ARDP); ○ = high-RDP diet (HRDP). Figure 1. View largeDownload slide Effect of ruminally degradable protein (RDP) on 15N-enrichment of ammonia N in dairy cows (means ±SE). • = adequate-RDP diet (ARDP); ○ = high-RDP diet (HRDP). Ruminal Sampling. Following thorough mixing of the isotope, a sample of the ruminal contents was taken (0 h sample), the ruminal contents were returned to the rumen, and ruminal samples were taken at 0.5, 1, 2, 4, 6, 8, 10, 14, 18, 24, and 30 h thereafter. Ruminal samples were taken from four locations in the rumen and the reticulum, composited, and analyzed for DM, OM, and 15N enrichment of the ammonia N and nonammonia N (NAN; Hristov and Ropp, 2003), and purine concentration in bacterial pellets. Purines were analyzed according to Zinn and Owens (1986) using the modified washing solution of Aharoni and Tagari (1991) and 0.6 M HClO4 (Makkar and Becker, 1999). Aliquots from the ruminal cheesecloth filtrates were immediately analyzed for pH, and processed for analysis of ammonia, VFA, reducing sugars, total free AA and peptides (Hristov et al., 1999), and polysaccharide-degrading (carboxymethylcellulase, amylase, and xylanase) and deaminative activities (Hristov et al., 2001b). Proteolytic activity in ruminal fluid was determined using 15N-labeled casein as a substrate according to Hristov et al. (2002). Ruminal samples were preserved and total protozoa counted as described (Hristov et al., 2001b). Chromium-EDTA (equivalent of 2.5 g of Cr/cow; Udén et al., 1980) was used as a ruminal liquid passage rate marker and was mixed with the ruminal contents at 0600 (during ruminal evacuation) on d 15 of each period. Ruminal samples were filtered through cheesecloth, centrifuged, and the supernatant fluids were analyzed for Cr concentration (Hristov and Ropp, 2003). Fractional outflow rate of the ruminal fluid phase was calculated as natural log-transformed Cr concentrations plotted vs. time. The volume of ruminal fluid was calculated as Cr dose divided by the antilog of the regression intercept (Cr concentration at 0 h). The pool size of ruminal ammonia N was calculated as: ruminal volume (L) × average ammonia N concentration (g/L). Milk Sampling. Total milk output was measured and milk was analyzed for fat, true protein, and milk urea nitrogen (MUN) during the last 7 d of each period (Washington DHIA, Burlington, WA). Following the 15N dose, cows were milked at 0 (background), 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 105, and 120 h. At each milking, milk weights were recorded and two milk samples were taken. One was used for analyses of milk fat, true protein, and MUN and another for analysis of 15N-enrichment of milk protein (Hristov and Ropp, 2003). Calculations. Ruminal ammonia (Figure 1), milk (Figure 2), and bacteria (Figure 3) 15N-enrichment (atom percent excess) curves were plotted vs. time and fitted to a three-parameter single exponential decay model, a five-parameter Weibull peak model, or to a four-parameter double exponential decay model (Sigma Plot 8.0 regression models library, SPSS Inc., Chicago, IL), respectively. Criteria for best fit were previously described (Hristov and Ropp, 2003). The average adjusted r2 (±SE) was 1.00 ± 0.002, 0.97 ± 0.018, and 0.96 ± 0.014 for ruminal ammonia N, bacterial N, and milk protein N, respectively. Areas under the predicted milk protein, ruminal ammonia, and bacterial 15N curves (15N atom percent excess × h) were computed using the trapezoidal rule (AREA.XFM transform, SigmaPlot 8.0). Proportions of milk protein N originating from ruminal bacterial and ammonia N and the proportion of bacterial N originating from ruminal ammonia N were derived based on the respective AUC (Nolan and Leng, 1974). Figure 2. View largeDownload slide Effect of ruminally degradable protein (RDP) on 15N-enrichment of milk protein in dairy cows (means ±SE). • = adequate-RDP diet (ARDP); ○ = high-RDP diet (HRDP). Figure 2. View largeDownload slide Effect of ruminally degradable protein (RDP) on 15N-enrichment of milk protein in dairy cows (means ±SE). • = adequate-RDP diet (ARDP); ○ = high-RDP diet (HRDP). Figure 3. View largeDownload slide Effect of ruminally degradable protein (RDP) on 15N-enrichment of bacterial N in dairy cows (means ±SE). • = adequate-RDP diet (ARDP); ○ = high-RDP diet (HRDP). Figure 3. View largeDownload slide Effect of ruminally degradable protein (RDP) on 15N-enrichment of bacterial N in dairy cows (means ±SE). • = adequate-RDP diet (ARDP); ○ = high-RDP diet (HRDP). The total flux, irreversible loss, and recycling rate of ruminal ammonia N were calculated from ammonia 15N-enrichment data, the 15N-ammonia AUC, and by difference according to Nolan and Leng (1974). The microbial N leaving the rumen that originated from ruminal ammonia N was calculated as microbial N flow × proportion of bacterial N originating from ammonia N. The proportion of the irreversible ammonia N loss incorporated into microbial protein leaving the rumen was calculated as follows: [(microbial N flow × proportion of bacterial N derived from ammonia N)/irreversible loss of ammonia N] × 100. The cumulative amount of 15N secreted in milk protein was calculated as milk output for each milking interval multiplied by the trichloroacetic acid-precipitable N concentration of milk (Hristov and Ropp, 2003) and by its 15N-enrichment. Data were presented as the percentage of 15N dosed (for each individual cow) and were fitted to a single rectangular two-parameter hyperbola model of the type: f = a ×x/(b + x) (PROC NLIN, SAS Inst., Inc., Cary, NC; Figure 4). In this case, a represented the theoretical maximum of 15N secreted with milk protein as a percentage of 15N dosed in the rumen. The proportion of the variance explained by the model (regression sums of squares/uncorrected total sums of squares) was 0.98 and 0.99 (ARDP and HRDP, respectively). Estimated maximal secretions for the two treatments were compared using the dummy variable regression technique (Hristov and Ropp, 2003). Figure 4. View largeDownload slide Effect of ruminally degradable protein (RDP) on cumulative secretion curves of 15N in milk protein in dairy cows expressed as percentage of 15N dosed intraruminally (measured and predicted values). Closed circles and solid line, adequate-RDP diet (ARDP), measured and predicted, respectively; Open circles and dashed line, high-RDP diet (HRDP), measured and predicted, respectively. Figure 4. View largeDownload slide Effect of ruminally degradable protein (RDP) on cumulative secretion curves of 15N in milk protein in dairy cows expressed as percentage of 15N dosed intraruminally (measured and predicted values). Closed circles and solid line, adequate-RDP diet (ARDP), measured and predicted, respectively; Open circles and dashed line, high-RDP diet (HRDP), measured and predicted, respectively. Nutrient Flow at the Duodenum and Total-Tract Digestibility Determinations Nutrient Flow. Flow and ruminal digestibility of DM, OM, NAN, NDF, and flow of microbial N at the duodenum were determined using the double-marker method of Faichney (1975). Lithium/CoEDTA and indigestible NDF were utilized as fluid and particulate phase markers, respectively. For details on flow markers dosing, sampling, and analyses see Hristov and Ropp (2003). Total-Tract Digestibility. Total-tract apparent digestibility of DM, OM, CP, and NDF was determined using acid-insoluble ash (Van Keulen and Young, 1977) as an internal digestibility marker. Sampling and analyses of feces were as described by Hristov and Ropp (2003). Other Analyses Diets, alfalfa hay, and triticale silage were sampled once each week, and concentrate feeds were sampled once per period. Samples were composited per period, dried at 60°C to constant weight in a forced-air oven, and analyzed for ash, acid-insoluble ash, N (Hristov et al., 2001a), NDF (Ankom 200 Fiber Analyzer, Ankom Technology), starch, ether extract, and lignin. A heat-stable amylase (α-amylase, EC No. 232-560-9, Sigma Chemical Co., St. Louis, MO) was used in the NDF analysis, but sodium sulfite was not used (Van Soest et al., 1991). Lignin (sulfuric acid lignin) was analyzed using the Daisy II Incubator (Ankom Technology). Starch was analyzed using a total starch analysis kit (Megazyme International Ireland Ltd., Wicklow, Ireland; McCleary et al., 1994), and ether extract was analyzed using Ankom XT 10 Extractor (Ankom Technology). Concentration of nonfibrous carbohydrates (NFC) in the diets was calculated as: NSC = 100 −(CP + NDF + ash + ether extract) (NRC, 2001). Available NDF (ANDF) was calculated as: ANDF = NDF −(lignin × 2.4) (CPM Dairy v. 2.0.23; Univ. of Pennsylvania, Kennett Square; Cornell Univ., Ithaca, NY; and William H. Miner Agric. Res. Inst., Chazy, NY). Ruminally undegradable protein concentration of the diets was determined according to Broderick (1987). Protein escaping ruminal degradation (RUP) was estimated for each diet ingredient assuming ruminal passage rate of 0.06 h−1. Dietary RUP was calculated based on protein undegradability of diet ingredients. Ammonia concentration was determined in fresh feces. Twenty milliliters of 0.5 M H2SO4 was added to 10 g of a composited fecal sample and the suspension was agitated at 120 rpm for 30 min at 25°C. Samples were first centrifuged at 17,400 ×g for 15 min at 4°C, 65% trichloroacetic acid was added to the supernatant to give a 5% final concentration, and the sample was re-centrifuged at 20,000 ×g for 15 min at 4°C. Ammonia concentration in the supernatant fraction was analyzed as for the ruminal fluid. Solubility of fecal N was determined as follows. Replicated fecal samples, 0.5 g dried and ground to pass a 1-mm sieve, were incubated with continuous agitation for 3 h in 125-mL Erlenmeyer flasks with 50 mL of borate-phosphate buffer (Licitra et al., 1996) and 1 mL of 10% (wt/vol) sodium azide solution. Samples were filtered through Whatman No. 54 filter paper using mild vacuum and residues were washed with cold distilled water. Filtrates, representing soluble fecal matter, were freeze-dried and analyzed for N. Total urine collection was performed during the last 4 d of each period and analyzed for N and allantoin concentration. Catheterization, sample preparation, and analyses were as described (Hristov and Ropp, 2003). On the last 2 d of each period, blood samples were taken from the jugular vein before (0 h) and 6 h after the morning feeding. Plasma was collected after centrifugation at 1,500 ×g for 40 min, frozen at −40°C, and later analyzed for urea N (PUN; urea nitrogen kit, Ct. No. 640-8; Sigma Diagnostics, St. Louis, MO). Statistical Analyses Intake, ruminal fermentation, duodenal nutrient flows, digestibility, and urine data were analyzed using ANOVA assuming a crossover design (SAS PROC MIXED). Ruminal fermentation data were averaged per period and cow. Statistical analysis of total protozoal counts was performed on log10-transformed data. The model used was:  \[Y_{ijkl}\ {=}\ {\mu}\ {+}\ G_{i}\ {+}\ C(G)_{ij}\ {+}\ P_{k}\ {+}\ D_{l}\ {+}\ e_{ijkl}\] [1] where μ is the overall mean, G, C, P, and D are group, cow, period, and treatment, and e is an error term under the usual assumptions for ANOVA (the error is distributed normally with mean = 0 and a constant variance). The term C(G)ij was included as a random effect. Statistical difference was declared at P < 0.05 and P < 0.12 was considered a tendency. Results Diets contained similar amounts of RUP, but differed in RDP and CP (Table 1). Estimated (NRC, 2001) MP concentrations were similar for both diets. Diets were formulated to provide 9 and 496 g/d of RDP in excess of requirements (NRC, 2001; ARDP and HRDP, respectively). Diets had similar concentrations of NDF and ANDF, but diet ARDP contained slightly more starch (+7.8%) and NFC (+7.4%) than diet HRDP. Ruminal ammonia concentration tended to be greater (P = 0.06) for HRDP compared with ARDP (Table 2). Diet did not affect ruminal pH, reducing sugars, total free AA, peptides, protozoa, or concentrations of total and major VFA. Concentrations of valerate and branched-chain VFA (BCVFA) were increased, or tended to be increased (P = 0.11, P = 0.04, and P = 0.07 for valerate, iso-butyrate, and iso-valerate, respectively) with HRDP compared with the ARDP diet. Polysaccharide-degrading, deaminative, and proteolytic activities, and fractional outflow rate of ruminal fluid were not affected by diet. Microbial N flow at the duodenum or the flow of MN formed from ammonia N and the efficiency of MPS in the rumen also were not statistically different between treatments, although MN flow was numerically greater (by 10%; P = 0.32) with HRDP vs. ARDP. Urinary excretion of allantoin tended to be increased (P = 0.08) with HRDP vs. ARDP. Table 2. Effects of ruminally degradable protein on ruminal fermentation in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  pH  6.41  6.45  0.098  0.58  Ammonia, mM  8.7  10.9  0.60  0.06      Ammonia N, gb  10.0  13.7  1.38  0.19  Reducing sugars, mM  4.5  4.4  0.75  0.95  Total free AA, mM  6.1  7.0  1.73  0.37  Peptides, mM  13.1  14.1  0.90  0.13  Total protozoa, × 104 cells/mLc  41.8 (5.59)  35.6 (5.54)  (0.067)  0.46  Volatile fatty acids, mM      Acetate  46.8  47.6  1.41  0.57      Propionate  19.4  19.6  0.42  0.77      iso-Butyrate  1.03  1.11  0.023  0.04      Butyrate  9.8  10.1  0.44  0.28      iso-Valerate  1.18  1.32  0.048  0.07      Valerate  1.22  1.31  0.032  0.11      Total VFA  79.4  81.0  2.31  0.45      Acetate:propionate  2.41  2.44  0.040  0.60  Polysaccharide-degrading activitiesd      CMCase  49.0  46.6  6.89  0.31      Xylanase  270.3  258.9  28.42  0.68      Amylase  42.5  34.8  6.03  0.35  Deaminative activitye  4.05  4.31  0.328  0.55  Proteolytic activityf  14.6  8.4  2.69  0.16  Liquid phase FOR, %/hg  13.3  12.8  0.95  0.18  MN flow, g/dh  230.4  253.5  18.80  0.32      MN from NH3 flow, g/di  95.2  110.6  9.08  0.32  MN, g/kg OMTDRj  16.2  17.5  0.75  0.33  Urinary allantoin, g/d  45.8  53.6  4.36  0.08    Dieta      Item  ARDP  HRDP  SE  P-value  pH  6.41  6.45  0.098  0.58  Ammonia, mM  8.7  10.9  0.60  0.06      Ammonia N, gb  10.0  13.7  1.38  0.19  Reducing sugars, mM  4.5  4.4  0.75  0.95  Total free AA, mM  6.1  7.0  1.73  0.37  Peptides, mM  13.1  14.1  0.90  0.13  Total protozoa, × 104 cells/mLc  41.8 (5.59)  35.6 (5.54)  (0.067)  0.46  Volatile fatty acids, mM      Acetate  46.8  47.6  1.41  0.57      Propionate  19.4  19.6  0.42  0.77      iso-Butyrate  1.03  1.11  0.023  0.04      Butyrate  9.8  10.1  0.44  0.28      iso-Valerate  1.18  1.32  0.048  0.07      Valerate  1.22  1.31  0.032  0.11      Total VFA  79.4  81.0  2.31  0.45      Acetate:propionate  2.41  2.44  0.040  0.60  Polysaccharide-degrading activitiesd      CMCase  49.0  46.6  6.89  0.31      Xylanase  270.3  258.9  28.42  0.68      Amylase  42.5  34.8  6.03  0.35  Deaminative activitye  4.05  4.31  0.328  0.55  Proteolytic activityf  14.6  8.4  2.69  0.16  Liquid phase FOR, %/hg  13.3  12.8  0.95  0.18  MN flow, g/dh  230.4  253.5  18.80  0.32      MN from NH3 flow, g/di  95.2  110.6  9.08  0.32  MN, g/kg OMTDRj  16.2  17.5  0.75  0.33  Urinary allantoin, g/d  45.8  53.6  4.36  0.08  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Ammonia N pool size (g) estimated based on Cr-EDTA kinetics and average ammonia concentration in the rumen (see Materials and Methods). c Values shown are means of actual counts. The values in parentheses are the means of log10 transformations of the data immediately above. d Expressed as nanomoles of reducing sugars as glucose released per milliliter of ruminal fluid per minute; CMCase = carboxymethyl-cellulase. e Expressed as micromoles of ammonia released from 1 mL of ruminal fluid/h. f Expressed as micrograms of N from 15N-casein released from 1 mL of ruminal fluid/min. g FOR = fractional outflow rate. h MN = microbial nitrogen. i Calculated as MN flow × proportion of bacterial N originating from ammonia N (area under the 15N curve data, Table 5). j OMTDR = OM truly digested in the rumen. View Large Table 2. Effects of ruminally degradable protein on ruminal fermentation in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  pH  6.41  6.45  0.098  0.58  Ammonia, mM  8.7  10.9  0.60  0.06      Ammonia N, gb  10.0  13.7  1.38  0.19  Reducing sugars, mM  4.5  4.4  0.75  0.95  Total free AA, mM  6.1  7.0  1.73  0.37  Peptides, mM  13.1  14.1  0.90  0.13  Total protozoa, × 104 cells/mLc  41.8 (5.59)  35.6 (5.54)  (0.067)  0.46  Volatile fatty acids, mM      Acetate  46.8  47.6  1.41  0.57      Propionate  19.4  19.6  0.42  0.77      iso-Butyrate  1.03  1.11  0.023  0.04      Butyrate  9.8  10.1  0.44  0.28      iso-Valerate  1.18  1.32  0.048  0.07      Valerate  1.22  1.31  0.032  0.11      Total VFA  79.4  81.0  2.31  0.45      Acetate:propionate  2.41  2.44  0.040  0.60  Polysaccharide-degrading activitiesd      CMCase  49.0  46.6  6.89  0.31      Xylanase  270.3  258.9  28.42  0.68      Amylase  42.5  34.8  6.03  0.35  Deaminative activitye  4.05  4.31  0.328  0.55  Proteolytic activityf  14.6  8.4  2.69  0.16  Liquid phase FOR, %/hg  13.3  12.8  0.95  0.18  MN flow, g/dh  230.4  253.5  18.80  0.32      MN from NH3 flow, g/di  95.2  110.6  9.08  0.32  MN, g/kg OMTDRj  16.2  17.5  0.75  0.33  Urinary allantoin, g/d  45.8  53.6  4.36  0.08    Dieta      Item  ARDP  HRDP  SE  P-value  pH  6.41  6.45  0.098  0.58  Ammonia, mM  8.7  10.9  0.60  0.06      Ammonia N, gb  10.0  13.7  1.38  0.19  Reducing sugars, mM  4.5  4.4  0.75  0.95  Total free AA, mM  6.1  7.0  1.73  0.37  Peptides, mM  13.1  14.1  0.90  0.13  Total protozoa, × 104 cells/mLc  41.8 (5.59)  35.6 (5.54)  (0.067)  0.46  Volatile fatty acids, mM      Acetate  46.8  47.6  1.41  0.57      Propionate  19.4  19.6  0.42  0.77      iso-Butyrate  1.03  1.11  0.023  0.04      Butyrate  9.8  10.1  0.44  0.28      iso-Valerate  1.18  1.32  0.048  0.07      Valerate  1.22  1.31  0.032  0.11      Total VFA  79.4  81.0  2.31  0.45      Acetate:propionate  2.41  2.44  0.040  0.60  Polysaccharide-degrading activitiesd      CMCase  49.0  46.6  6.89  0.31      Xylanase  270.3  258.9  28.42  0.68      Amylase  42.5  34.8  6.03  0.35  Deaminative activitye  4.05  4.31  0.328  0.55  Proteolytic activityf  14.6  8.4  2.69  0.16  Liquid phase FOR, %/hg  13.3  12.8  0.95  0.18  MN flow, g/dh  230.4  253.5  18.80  0.32      MN from NH3 flow, g/di  95.2  110.6  9.08  0.32  MN, g/kg OMTDRj  16.2  17.5  0.75  0.33  Urinary allantoin, g/d  45.8  53.6  4.36  0.08  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Ammonia N pool size (g) estimated based on Cr-EDTA kinetics and average ammonia concentration in the rumen (see Materials and Methods). c Values shown are means of actual counts. The values in parentheses are the means of log10 transformations of the data immediately above. d Expressed as nanomoles of reducing sugars as glucose released per milliliter of ruminal fluid per minute; CMCase = carboxymethyl-cellulase. e Expressed as micromoles of ammonia released from 1 mL of ruminal fluid/h. f Expressed as micrograms of N from 15N-casein released from 1 mL of ruminal fluid/min. g FOR = fractional outflow rate. h MN = microbial nitrogen. i Calculated as MN flow × proportion of bacterial N originating from ammonia N (area under the 15N curve data, Table 5). j OMTDR = OM truly digested in the rumen. View Large Cows on the HRDP diet consumed more N (P = 0.003; Table 3) and slightly more NDF (2.5%, P = 0.03) and tended to consume more (P = 0.09) DM and OM than the cows on the ARDP diet (Table 4). Ruminal apparent or true digestibility of DM, OM, NDF, and N did not differ significantly between diets. Total-tract apparent digestibility of DM, OM, and NDF was not affected by treatment. Total-tract digestibility of N was greater (P = 0.02) with HRDP than with the ARDP diet. Fecal ammonia concentration, solubility of fecal N, and C:N ratio of fecal DM were not affected by diet. Table 3. Effects of ruminally degradable protein on nitrogen losses and plasma (PUN) and milk (MUN) urea N concentrations in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  N intake, g/d  595  695  2.3  0.003  Milk N yield, g/db  110  109  10.3  0.93      % of intake  18.4  15.7  1.21  0.01  Total N losses, g/d  437  509  17.6  0.06      % of intake  73.4  73.3  2.85  0.97  Urine N, g/d  252  321  13.8  0.07      % of total excreted  57.7  62.7  1.75  0.12      % of intake  42.5  46.3  2.95  0.38  Fecal N, g/d  185  188  1.1  0.48      % of total excreted  42.3  37.3  1.75  0.12      % of intake  31.0  27.0  0.68  0.02  PUN, mg/100 mL  14.7  19.4  1.68  0.002  MUN, mg/100 mL  13.1  15.8  1.62  0.01    Dieta      Item  ARDP  HRDP  SE  P-value  N intake, g/d  595  695  2.3  0.003  Milk N yield, g/db  110  109  10.3  0.93      % of intake  18.4  15.7  1.21  0.01  Total N losses, g/d  437  509  17.6  0.06      % of intake  73.4  73.3  2.85  0.97  Urine N, g/d  252  321  13.8  0.07      % of total excreted  57.7  62.7  1.75  0.12      % of intake  42.5  46.3  2.95  0.38  Fecal N, g/d  185  188  1.1  0.48      % of total excreted  42.3  37.3  1.75  0.12      % of intake  31.0  27.0  0.68  0.02  PUN, mg/100 mL  14.7  19.4  1.68  0.002  MUN, mg/100 mL  13.1  15.8  1.62  0.01  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Milk N yield = milk true protein yield/6.38. View Large Table 3. Effects of ruminally degradable protein on nitrogen losses and plasma (PUN) and milk (MUN) urea N concentrations in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  N intake, g/d  595  695  2.3  0.003  Milk N yield, g/db  110  109  10.3  0.93      % of intake  18.4  15.7  1.21  0.01  Total N losses, g/d  437  509  17.6  0.06      % of intake  73.4  73.3  2.85  0.97  Urine N, g/d  252  321  13.8  0.07      % of total excreted  57.7  62.7  1.75  0.12      % of intake  42.5  46.3  2.95  0.38  Fecal N, g/d  185  188  1.1  0.48      % of total excreted  42.3  37.3  1.75  0.12      % of intake  31.0  27.0  0.68  0.02  PUN, mg/100 mL  14.7  19.4  1.68  0.002  MUN, mg/100 mL  13.1  15.8  1.62  0.01    Dieta      Item  ARDP  HRDP  SE  P-value  N intake, g/d  595  695  2.3  0.003  Milk N yield, g/db  110  109  10.3  0.93      % of intake  18.4  15.7  1.21  0.01  Total N losses, g/d  437  509  17.6  0.06      % of intake  73.4  73.3  2.85  0.97  Urine N, g/d  252  321  13.8  0.07      % of total excreted  57.7  62.7  1.75  0.12      % of intake  42.5  46.3  2.95  0.38  Fecal N, g/d  185  188  1.1  0.48      % of total excreted  42.3  37.3  1.75  0.12      % of intake  31.0  27.0  0.68  0.02  PUN, mg/100 mL  14.7  19.4  1.68  0.002  MUN, mg/100 mL  13.1  15.8  1.62  0.01  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Milk N yield = milk true protein yield/6.38. View Large Table 4. Effects of ruminally degradable protein on nutrient intakes and digestibility in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  Intake, kg/d      DM  23.5  23.8  0.85  0.09      OM  21.5  21.8  0.78  0.09      NDF  8.0  8.2  0.29  0.03  Digestibility, %      Rumen, apparent          DM  52.7  51.4  2.32  0.64              DM, true  65.4  65.1  2.99  0.89          OM  54.5  53.3  2.37  0.64              OM, true  66.4  66.0  2.99  0.83      N, true  71.8  77.1  4.99  0.29          NDF  54.6  51.8  0.97  0.12      Total tract, apparent          DM  71.7  71.7  1.22  0.97          OM  73.3  73.4  1.25  0.72          N  69.0  73.0  0.68  0.02          NDF  58.3  57.2  3.08  0.39  Fecal ammonia, mg/g DM  0.84  0.89  0.083  0.67  Soluble fecal N, % of total N  31.5  27.6  1.86  0.14  Fecal C:N ratio  16.3  16.1  0.49  0.46    Dieta      Item  ARDP  HRDP  SE  P-value  Intake, kg/d      DM  23.5  23.8  0.85  0.09      OM  21.5  21.8  0.78  0.09      NDF  8.0  8.2  0.29  0.03  Digestibility, %      Rumen, apparent          DM  52.7  51.4  2.32  0.64              DM, true  65.4  65.1  2.99  0.89          OM  54.5  53.3  2.37  0.64              OM, true  66.4  66.0  2.99  0.83      N, true  71.8  77.1  4.99  0.29          NDF  54.6  51.8  0.97  0.12      Total tract, apparent          DM  71.7  71.7  1.22  0.97          OM  73.3  73.4  1.25  0.72          N  69.0  73.0  0.68  0.02          NDF  58.3  57.2  3.08  0.39  Fecal ammonia, mg/g DM  0.84  0.89  0.083  0.67  Soluble fecal N, % of total N  31.5  27.6  1.86  0.14  Fecal C:N ratio  16.3  16.1  0.49  0.46  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. View Large Table 4. Effects of ruminally degradable protein on nutrient intakes and digestibility in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  Intake, kg/d      DM  23.5  23.8  0.85  0.09      OM  21.5  21.8  0.78  0.09      NDF  8.0  8.2  0.29  0.03  Digestibility, %      Rumen, apparent          DM  52.7  51.4  2.32  0.64              DM, true  65.4  65.1  2.99  0.89          OM  54.5  53.3  2.37  0.64              OM, true  66.4  66.0  2.99  0.83      N, true  71.8  77.1  4.99  0.29          NDF  54.6  51.8  0.97  0.12      Total tract, apparent          DM  71.7  71.7  1.22  0.97          OM  73.3  73.4  1.25  0.72          N  69.0  73.0  0.68  0.02          NDF  58.3  57.2  3.08  0.39  Fecal ammonia, mg/g DM  0.84  0.89  0.083  0.67  Soluble fecal N, % of total N  31.5  27.6  1.86  0.14  Fecal C:N ratio  16.3  16.1  0.49  0.46    Dieta      Item  ARDP  HRDP  SE  P-value  Intake, kg/d      DM  23.5  23.8  0.85  0.09      OM  21.5  21.8  0.78  0.09      NDF  8.0  8.2  0.29  0.03  Digestibility, %      Rumen, apparent          DM  52.7  51.4  2.32  0.64              DM, true  65.4  65.1  2.99  0.89          OM  54.5  53.3  2.37  0.64              OM, true  66.4  66.0  2.99  0.83      N, true  71.8  77.1  4.99  0.29          NDF  54.6  51.8  0.97  0.12      Total tract, apparent          DM  71.7  71.7  1.22  0.97          OM  73.3  73.4  1.25  0.72          N  69.0  73.0  0.68  0.02          NDF  58.3  57.2  3.08  0.39  Fecal ammonia, mg/g DM  0.84  0.89  0.083  0.67  Soluble fecal N, % of total N  31.5  27.6  1.86  0.14  Fecal C:N ratio  16.3  16.1  0.49  0.46  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. View Large Table 5. Effects of ruminally degradable protein on 15N enrichment of N pools and 15N transformations in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  AUC, ruminal ammonia Nb  4.22  3.67  0.235  0.24  AUC, ruminal bacterial N  1.73  1.61  0.049  0.22  AUC, milk protein N  1.05  0.96  0.014  0.02  Bacterial N from ammonia N, %c  41.3  43.7  1.68  0.35  Milk protein N from bacterial N, %c  60.8  60.6  1.89  0.94  Milk protein N from ammonia N, %c  25.1  26.4  1.23  0.53  Irreversible loss of ruminal ammonia N, g N/d  247  284  14.2  0.21  % of N intake  41.4  41.0  2.30  0.89  Ruminal ammonia N flux, g/d  366  461  49.9  0.31  Recycled ammonia N, g/d  118  177  36.9  0.38  Utilization of ruminal ammonia N for microbial protein synthesis, %d  38.8  38.8  2.60  0.88  Regression analysis, cumulative milk protein 15N secretion (estimate ± approx. SE)e  Maximum 15N secreted,  17.3 ± 1.06  15.8 ± 1.18    0.35  % of dosedf Comparison of predicted lines        0.003    Dieta      Item  ARDP  HRDP  SE  P-value  AUC, ruminal ammonia Nb  4.22  3.67  0.235  0.24  AUC, ruminal bacterial N  1.73  1.61  0.049  0.22  AUC, milk protein N  1.05  0.96  0.014  0.02  Bacterial N from ammonia N, %c  41.3  43.7  1.68  0.35  Milk protein N from bacterial N, %c  60.8  60.6  1.89  0.94  Milk protein N from ammonia N, %c  25.1  26.4  1.23  0.53  Irreversible loss of ruminal ammonia N, g N/d  247  284  14.2  0.21  % of N intake  41.4  41.0  2.30  0.89  Ruminal ammonia N flux, g/d  366  461  49.9  0.31  Recycled ammonia N, g/d  118  177  36.9  0.38  Utilization of ruminal ammonia N for microbial protein synthesis, %d  38.8  38.8  2.60  0.88  Regression analysis, cumulative milk protein 15N secretion (estimate ± approx. SE)e  Maximum 15N secreted,  17.3 ± 1.06  15.8 ± 1.18    0.35  % of dosedf Comparison of predicted lines        0.003  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Area under the 15N curve, APE × h. c Calculated as (AUCrumen bacteria/AUCrumen ammonia) × 100; (AUC milk protein/AUC rumen bacteria) × 100; or (AUC milk protein/AUC rumen ammonia) × 100, respectively. d Proportion of the irreversible loss of ammonia N leaving the rumen as microbial N. Calculated as [(MN flow × proportion of bacterial N derived from ammonia N)/irreversible loss of ammonia N] × 100. e15 Nitrogen secretion in milk (mg) as a percentage of 15N infused in the rumen (mg) fitted to a single rectangular two-parameter hyperbola model (Figure 4). f Theoretical maximum. View Large Table 5. Effects of ruminally degradable protein on 15N enrichment of N pools and 15N transformations in dairy cows (least squares means and associated SE)   Dieta      Item  ARDP  HRDP  SE  P-value  AUC, ruminal ammonia Nb  4.22  3.67  0.235  0.24  AUC, ruminal bacterial N  1.73  1.61  0.049  0.22  AUC, milk protein N  1.05  0.96  0.014  0.02  Bacterial N from ammonia N, %c  41.3  43.7  1.68  0.35  Milk protein N from bacterial N, %c  60.8  60.6  1.89  0.94  Milk protein N from ammonia N, %c  25.1  26.4  1.23  0.53  Irreversible loss of ruminal ammonia N, g N/d  247  284  14.2  0.21  % of N intake  41.4  41.0  2.30  0.89  Ruminal ammonia N flux, g/d  366  461  49.9  0.31  Recycled ammonia N, g/d  118  177  36.9  0.38  Utilization of ruminal ammonia N for microbial protein synthesis, %d  38.8  38.8  2.60  0.88  Regression analysis, cumulative milk protein 15N secretion (estimate ± approx. SE)e  Maximum 15N secreted,  17.3 ± 1.06  15.8 ± 1.18    0.35  % of dosedf Comparison of predicted lines        0.003    Dieta      Item  ARDP  HRDP  SE  P-value  AUC, ruminal ammonia Nb  4.22  3.67  0.235  0.24  AUC, ruminal bacterial N  1.73  1.61  0.049  0.22  AUC, milk protein N  1.05  0.96  0.014  0.02  Bacterial N from ammonia N, %c  41.3  43.7  1.68  0.35  Milk protein N from bacterial N, %c  60.8  60.6  1.89  0.94  Milk protein N from ammonia N, %c  25.1  26.4  1.23  0.53  Irreversible loss of ruminal ammonia N, g N/d  247  284  14.2  0.21  % of N intake  41.4  41.0  2.30  0.89  Ruminal ammonia N flux, g/d  366  461  49.9  0.31  Recycled ammonia N, g/d  118  177  36.9  0.38  Utilization of ruminal ammonia N for microbial protein synthesis, %d  38.8  38.8  2.60  0.88  Regression analysis, cumulative milk protein 15N secretion (estimate ± approx. SE)e  Maximum 15N secreted,  17.3 ± 1.06  15.8 ± 1.18    0.35  % of dosedf Comparison of predicted lines        0.003  a ARDP = adequate ruminally degradable protein (RDP) diet; HRDP = high-RDP diet. b Area under the 15N curve, APE × h. c Calculated as (AUCrumen bacteria/AUCrumen ammonia) × 100; (AUC milk protein/AUC rumen bacteria) × 100; or (AUC milk protein/AUC rumen ammonia) × 100, respectively. d Proportion of the irreversible loss of ammonia N leaving the rumen as microbial N. Calculated as [(MN flow × proportion of bacterial N derived from ammonia N)/irreversible loss of ammonia N] × 100. e15 Nitrogen secretion in milk (mg) as a percentage of 15N infused in the rumen (mg) fitted to a single rectangular two-parameter hyperbola model (Figure 4). f Theoretical maximum. View Large Cows were in late lactation and produced 22.7 and 23.2 kg of milk daily (ARDP and HRDP, respectively; SE = 1.74; P = 0.64). Fat-corrected milk yield and milk fat, lactose, and true protein (3.08 and 3.01%, respectively) concentrations did not differ (P = 0.53, P = 0.62, P = 0.74, and P = 0.32, respectively) between the diets. As a proportion of intake, ARDP cows produced more (P = 0.01) milk N than the HRDP cows (Table 3). Overall, cows fed the HRDP diet tended to excrete more N (16%; P = 0.06) in feces plus urine than the ARDP cows, but as proportion of N intake, N losses were similar between the two diets. Excretion of urinary N tended to be greater (P = 0.07) with the HRDP than with the ARDP diet. Total N losses with the feces were similar between the diets. As proportion of intake, fecal N losses were greater (P = 0.02) with ARDP than with the HRDP diet. Plasma urea N and MUN concentrations were greater (by 32 and 21%; P = 0.002 and P = 0.01, respectively) for cows fed the HRDP diet. Ruminal ammonia and bacterial 15N AUC did not differ between diets (Table 5). The milk protein 15N AUC was greater (P = 0.02) for ARDP than for the HRDP diet (Figure 2). The estimates for bacterial N originating from ammonia N in the rumen (mean of 42.5%) and milk protein N originating from bacterial (mean of 60.7%) or ammonia N (mean of 25.8%) were similar between the two diets. The irreversible N loss from the ruminal ammonia N pool averaged 41.2% of N intake and was not affected by treatment. Estimated flux, recycling, and use of ammonia N for MPS in the rumen also did not differ between diets. The theoretical maximum 15N secreted in milk protein (as proportion of ruminal dose) was similar for both diets, but the overall secretion of 15N in milk protein during the course of sampling was greater (P = 0.003) for the ARDP than for the HRDP diet (Figure 4). Discussion Due to differences in protein degradation between SBM and SoyPass, determined and predicted RUP concentrations (NRC, 2001; 5.9% of dietary DM for both ARDP and HRDP diets) were similar between the two diets. Lignosulfonate treatment has decreased ruminal degradation of a number of protein feeds (Waltz and Stern, 1989; McAllister et al., 1993; Keyserlingk et al., 2000). With similar RUP concentration and predicted microbial protein flows (1,969 and 1,975 g/d for ARDP and HRDP, respectively), the two diets in this trial had similar estimated MP concentrations (NRC, 2001). Measured duodenal NAN flows were also not different between treatments (412 and 414 g/d for ARDP and HRDP, respectively; SE = 29.5, P = 0.97). With few exceptions, most studies investigating effects of dietary CP level on ruminal fermentation and production of dairy cows differed in RUP and MP/AA supply: Tomlinson et al., 1994; Powers et al., 1995; Reynal and Broderick, 2003 (RUP and/or MP were estimated from published diet composition using NRC, 2001), and Komaragiri and Erdman, 1997; Kröber et al., 2000; Haig et al., 2002 (RUP and/or MP reported by the authors). In several studies, dietary RDP supply varied, whereas cows were fed diets containing similar levels of MP or AA available from the intestine (Armentano et al., 1993; Frank et al., 2002; Davidson et al., 2003). The concentration of ammonia in the rumen is a function of both rate of ruminal N degradation and concentration of RDP above microbial needs and the amount of dietary energy available to the ruminal microorganisms; in most feeding systems, MPS is assumed to be energy-dependent (NKJ Protein Group, 1985; Tamminga et al., 1994; NRC, 2001). The addition of ruminally available N (from SBM) with the HRDP diet resulted in a 25% increase in ruminal ammonia concentration in this experiment. Similarly, Mansfield et al. (1994), Calsamiglia et al. (1995), and Stanford et al. (1995) reported that ruminal ammonia concentrations were greater with untreated SBM than with lignosulfonate-treated protein supplements. Increasing CP concentration or degradation of dietary protein usually results in increased ammonia concentrations in the rumen (Armentano et al., 1993; Olmos Colmenero and Broderick, 2003; Davidson et al., 2003). Concentrations of iso-butyrate and iso-valerate were greater with the HRDP diet in our study. In the rumen, BCVFA are derived from branched-chain AA (Wolin et al., 1997). Increased BCVFA was reported with increasing dietary CP from SBM (Broderick, 1986) or alfalfa silage (Hristov and Broderick, 1996). Windschitl and Stern (1988a,b) and Calsamiglia et al. (1995) did not find an effect of lignosulfonate-treated SBM on BCVFA concentrations in vitro or in vivo, but Calsamiglia et al. (1995) reported that flow from continuous culture fermentors of the branched-chain AA leucine, isoleucine, and valine was greater with lignosulfonate-treated compared with untreated SBM. Blood plasma concentration of isoleucine was greater for a high- vs. a low-CP diet in a study by Davidson et al. (2003). Levels of ruminally fermentable energy, as indicated by the dietary concentration of NSP and ANDF, were similar between the diets in the present experiment. In these conditions, the excess RDP in the HRDP diet did not produce a statistically significant response in MPS; however, both duodenal sampling and urinary excretion of allantoin (a 17% increase) indicated some numerical increase in MPS with the HRDP diet. Broderick (2003) reported that excretion of urinary purine derivatives increased as dietary CP was increased from 15.1 to 16.7% of diet DM, but no further increase was noted with an 18.4% CP diet. Haig et al. (2002) found urinary purine derivatives excretion increased quadratically as RDP (expressed as soluble intake N) concentration of the diet increased. Crude protein concentration or degradation most often does not affect microbial N flow (Windschitl and Stern, 1988a; Calsamiglia et al., 1995; Cunningham et al., 1996). In some studies, MPS decreased when protein supplements of low ruminal degradability were fed (Windschitl and Stern, 1988b). If the diet provides the rumen with sufficient amounts of degradable protein/ammonia N, energy intake is the primary factor explaining the variability in MPS (Clark et al., 1992; Oldick et al., 1999). The study by Broderick (2003) reported a greater effect of dietary energy (rolled high-moisture shelled corn) vs. CP (SBM) on MPS (as urinary purine derivatives excretion). Although sufficient in ruminally available energy (as NFC), the excess RDP provided with the HRDP diet in our study was not utilized to a significant extent by the ruminal microorganisms for MPS. Diets with elevated CP concentration usually have greater apparent N digestibility, partially due to dilution of metabolic fecal N and partially due to increased intake of more digestible feeds (Broderick, 2003). Inclusion in the diet of ruminally resistant proteins also can decrease overall DM digestibility compared with SBM-based diets (Reynal and Broderick, 2003). The lower N intake with the ARDP diet and increased urinary N losses with the HRDP diet in this trial resulted in proportionally greater fecal N excretion with the ARDP than with the HRDP diet. Increasing solubility of dietary N/RDP content of the diet decreased fecal N excretion (respectively increased N digestibility) in a quadratic manner in the study by Haig et al. (2002), but had no effect on fecal N loss in early-lactation cows (Davidson et al., 2003). The efficiency of use of dietary N, for milk protein production although low, was greater with the ARDP vs. the HRDP diet in the present experiment. Frank et al. (2002) and Broderick (2003) clearly demonstrated the negative effect of dietary CP on the efficiency of N use for milk protein synthesis in dairy cows. A meta-analysis of a large data set confirmed the negative relationship between dietary CP and milk N efficiency (Hristov et al., 2004b). At similar CP levels, however, protein degradability did not affect milk N efficiency (Haig et al., 2002; Davidson et al., 2003). Increased loss of N in urine is the most common effect observed with high-CP/RDP diets and the observed tendency for increased absolute and proportional losses of N with the HRDP diet in the present trial corresponds to reports by Kröber et al. (2000), Castillo et al. (2001), and Leonardi et al. (2003). Kebreab et al. (2002) reported a greater and exponential effect of N intake on urinary N with limited effects on fecal or milk N excretion. Using a combination of prediction equations (urine volume) and actual analyses (urine composition), de Boer et al. (2002) demonstrated the importance of the ruminal N balance (OEB; Tamminga et al., 1994) in decreasing N losses by dairy cows; increasing OEB from 0 (maximal use of RDP) to 1,000 g/cow daily resulted in linear increase in urinary N excretion. In another Dutch study, decreasing OEB to 0.4 kg/d resulted in a steady decrease in N loss (Berentsen and Giesen, 1996). In the present trial, the effect on urinary N corresponded to a decreased PUN and MUN with the ARDP diet. Concentration of MUN has been directly related to concentration of ruminal ammonia (Broderick and Clayton, 1997), PUN (Rooke and Thomas, 1985), and urinary urea N secretion (Ciszuk and Gebregziabher, 1994); MUN (Bach et al., 2000; Kröber et al., 2000; Broderick, 2003) and PUN (Christensen et al., 1993; Metcalf et al., 1996; Davidson et al., 2003) have been shown repeatedly to correlate positively to the level of dietary CP or RDP. The different RDP levels did not produce differences in fecal composition (ammonia, soluble N concentrations, C:N ratio) in the present experiment. Although urine is a major source of N in manure, decreased N concentration in feces can contribute to the production of “environmentally friendly” manure. Ruminal fermentation can have a dramatic effect on C:N ratio in feces (Hristov et al., 2003), but RDP in this experiment did not affect the fecal C:N ratio. Level of CP in the diet also has been shown to affect ammonia emissions from dairy operations. Külling et al. (2001) reported a 0.7-fold decrease in ammonia emissions as N intake was decreased; Frank et al. (2002) found on average a 270% decrease in ammonia release to air from manure recovered from cows fed 14 vs. 19% CP diets. Average proportions of bacterial N formed from ruminal ammonia N and milk protein formed from ammonia and bacterial N in this study were 43, 26, and 61%, respectively. Ammonia is a major source of N for the ruminal bacteria (particularly cellulolytic species; Russell et al., 1992) and bacteria form from 38% (Hristov et al., 2003) to 70 to 80% (Oldham et al., 1980; Leng and Nolan, 1984; Koenig et al., 2000) of their N from ammonia N. As a percentage of N intake, the irreversible loss of N from the ammonia N pool can be as low as 36% (Hristov et al., 2003) or as high as 88% (Oldham et al., 1980). Siddons et al. (1985) reported that 32 and 66% of the irreversible loss of ruminal ammonia in sheep was through incorporation into microbial N (grass silage and dried grass, respectively). Thus, depending on the diet and type of animal, a significant proportion of the irreversible loss of ruminal ammonia is due to absorption. Our data indicate that 39% of the irreversible ammonia N loss was to microbial N leaving the rumen, and 61% was due to ammonia absorption or outflow with the ruminal fluid phase. On average, 39% of milk proteins were synthesized from nonbacterial N sources. Our data showed average NAN flow to the duodenum of 413 g/d, of which microbial N flow was 242 g/d. If NRC (2001) efficiency coefficients are used (0.64 and 0.80 for microbial and RUP N, respectively), these flows represent 155 and 137 g/d of MP-N from microbial and nonmicrobial sources, or 53 and 47% of the total, respectively. The isotope data (Table 5) indicate that 67 and 43 g/d milk protein N output from bacterial and RUP sources, respectively; thus, 43 and 31% of the estimated MP flow (from bacterial protein and RUP, respectively) were used for synthesis of milk proteins. Milk proteins are synthesized primarily from blood free amino acids, and blood flow dictates the amount of AA supplied to the mammary gland (Linzell, 1974). Therefore, the above coefficients may represent the demand for AA by the mammary gland, considering the stage of lactation and level of production of the experimental cows, but differences also could suggest differences in use of MP from bacterial and ruminally undegraded protein. Raggio et al. (2003) demonstrated that the efficiency of use of certain AA for milk protein synthesis decreased linearly with increasing MP supply. Some authors suggested lower efficiency of use of microbial AA (due to an imbalanced AA profile) compared with casein (Cant et al., 1999). Less of the dosed ammonia-15N was recovered in milk protein with the HRDP than with the ARDP diet, suggesting poorer efficiency of use of ruminal ammonia N for milk protein synthesis when RDP exceeds requirements. Similarly, Holthausen (2002) found lower recovery of i.v. dosed 15N-urea into milk protein of dairy cows receiving a urea-supplemented diet (2.3% urea, DM basis) compared with the unsupplemented control diet. Milk urea N concentration and urinary N excretion also were elevated with the urea diet compared with the control. Our approach in estimating the proportion of 15N-ammonia used for milk protein synthesis is based on the assumption that most of the ruminal ammonia that is not used for synthesis of microbial protein in the rumen will be detoxified in the liver and excreted as urea in the urine and only a small proportion will be used for synthesis of nonessential AA, which can be used for various purposes and may or may not be eventually incorporated into milk proteins. Intravenous infusion of 15NH4Cl in sheep showed that less than 4% of the net 15N transfer across the liver was as glutamate (Lobley et al., 1995) and 80 to 90% of the 15N infused appeared in urea (Lobley et al., 1996). Thus, if not captured as microbial protein in the rumen, most of the ammonia N absorbed through the ruminal wall would be used for ureagenesis by the liver. 15Nitrogen enrichment of bacterial N in the rumen decreased rapidly after dosing to 5% of the 0-h values within 30 h (Figure 3). Milk protein 15N-enrichment also decreased rapidly within 100 to 110 h after dosing of the marker (or approximately 70 h after the decrease in bacterial enrichment; Figure 2). Thus, the regression-derived theoretical maximum of 15N secreted in milk protein will closely represent the true amount of ruminal ammonia-15N transferred into milk protein via microbial protein N. However, N recycled from the tissues through various routes into milk protein will continue for a comparatively long period. Across treatments, the recovery of ruminal ammonia-15N in milk protein in the current study (as proportion of 15N infused in the rumen) was similar to our previous estimates (12 to 14%; Hristov and Ropp, 2003). Recalculation of the data by Petri et al. (1988) showed that in the lactating goat, from 10.3 to 14.3% of the irreversible loss of ruminal ammonia N was recovered in milk protein via microbial protein. 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TI - Effect of dietary crude protein level and degradability on ruminal fermentation and nitrogen utilization in lactating dairy cows
JO - Journal of Animal Science
DO - 10.2527/2004.82113219x
DA - 2004-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/effect-of-dietary-crude-protein-level-and-degradability-on-ruminal-pXdoW0zIBm
SP - 3219
EP - 3229
VL - 82
IS - 11
DP - DeepDyve
ER -