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J. Anim. Sci. 2005. 83:408-421
© 2005 American Society of Animal Science

ANIMAL NUTRITION

Effect of carbohydrate source on ammonia utilization in lactating dairy cows1

A. N. Hristov2, J. K. Ropp, K. L. Grandeen, S. Abedi, R. P. Etter, A. Melgar and A. E. Foley

Department of Animal and Veterinary Science, University of Idaho, Moscow 83844-2330


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
This study was conducted to investigate the effect of dextrose, starch, NDF, and a carbohydrate (CHO) mix on utilization of ruminal ammonia in dairy cows. Four ruminally and duodenally cannulated Holstein cows (BW = 788 ± 31 kg; 217 ± 35 d in milk) were allocated to four treatments in a 4 x 4 Latin square design trial. Cows were fed an all alfalfa diet at 12-h intervals (DMI = 22.2 ± 0.25 kg/d). Treatments were control, white oat fiber (NDF); corn dextrose (GLU); cornstarch (STA); and a CHO mix (25% of each): apple pectin, GLU, STA, and NDF (MIX). Carbohydrates were introduced intraruminally during feeding at 20% of dietary DMI. Ruminal ammonia was labeled with 15N. Ruminal pH was the highest for NDF followed by STA and MIX and GLU (P < 0.001). Ruminal ammonia concentration and pool size were decreased by GLU and STA compared with NDF (P < 0.001 and P = 0.03, respectively). Acetate, isobutyrate, isovalerate, and total VFA concentration in the rumen were decreased (P = 0.009 to 0.001), and butyrate was increased (P < 0.001) by GLU compared with the other CHO. Microbial N flow to the duodenum was decreased (P < 0.05) by NDF compared with the other CHO, and the flow of microbial N formed from ammonia was greater for STA compared with GLU and NDF (P = 0.04 and 0.03, respectively). Urinary N loss was decreased (P = 0.05) by GLU and STA, but overall (feces plus urine) N losses were not affected (P = 0.73) by treatment. Milk urea concentration was lowered by GLU and STA compared with NDF and MIX (P = 0.002). The proportion of bacterial N synthesized from ammonia in the rumen was greater with STA than with NDF and MIX and was least for GLU (P = 0.02). Irreversible ammonia loss and flux were lower (P = 0.09 and 0.02, respectively) for GLU than for STA and NDF. As a percentage of the dose given, cumulative secretion of 15N ammonia in milk protein was greater for STA than for GLU or NDF (P = 0.01 and 0.001, respectively). This experiment demonstrated that provision of readily fermentable energy can decrease ammonia concentrations in the rumen through decreased ammonia production (GLU), or through enhanced uptake of ammonia for microbial protein synthesis (STA). Rapidly fermentable energy in the rumen decreased ammonia production and flux, but the overall efficiency of ammonia utilization for milk protein synthesis was only increased by enhancing ruminal microbial ammonia uptake.

Key Words: Ammonia Utilization • Carbohydrate • Dairy Cows


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 
Ammonia concentration in the rumen can vary greatly depending on diet, time of feeding and feeding frequency, animal, and other factors. Some variants may cause a decrease in the efficiency of microbial capture of ammonia and eventually, in N wastage. The extent to which ammonia is utilized in the rumen depends primarily on the rate of release and the balance of carbohydrate (CHO) and N availability. Carbohydrate availability determines the rate of microbial growth in the rumen (Isaacson et al., 1975Go; Strobel and Russell, 1986Go; Hoover and Stokes, 1991Go) and efficiency of ruminal ammonia utilization (Newbold and Rust, 1992Go; Hristov et al., 1997Go; Heldt et al., 1999Go). If energy is limiting, ruminal microorganisms degrade feed proteins to ammonia (Russell et al., 1983Go), and microbial ammonia uptake is suppressed (Nocek and Russell, 1988Go; Hristov et al., 1997Go). Thus, CHO availability for ruminal fermentation is a key factor for improving the efficiency of ruminal ammonia and overall dietary N utilization in ruminants. Studies have investigated fermentation and production effects of addition of starch and/or sugars to the diet of lactating dairy cows (Broderick et al., 2000Go; McCormick et al., 2001Go; Sannes et al., 2002Go), but CHO sources were added to complete diets, rich in available energy, and in proportions not exceeding 3 to 7.5% of dietary DM.

We hypothesized that the provision of ruminally fermentable energy can enhance ammonia utilization by the ruminal microflora and transfer of ammonia N into milk protein in dairy cows. Thus, the objective of this experiment was to investigate the effects of adding CHO with different degradation rates to an energy-deficient basal diet on ruminal fermentation, microbial protein flow, and ammonia and overall N utilization in lactating dairy cows.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Implications
 Literature Cited
 
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 (BW = 788 ± 31 kg; 217 ± 35 d in milk at the beginning of the trial) were cared for according to the guidelines of the University of Idaho Animal Care and Use Committee and were subjected to the experimental treatments in a Latin square design. Treatments were as follows: control (NDF; Canadian Harvest oat fiber 200–150; Opta Food Ingredients, Inc., Bedford, MA); corn dextrose (GLU; CornProducts Int., Bedford Park, IL); cornstarch (STA; Roquette America, Inc., Keokuk, IA); and a CHO mix (25% of each) comprising apple pectin (Amcan Industries, Inc., Elmsford, NY), GLU, STA, and NDF (MIX). Corn dextrose and STA were selected to provide rapid or slower (respectively) release of fermentable energy in the rumen. Oat fiber was used as a negative control. The combination treatment (MIX) was designed to provide a steady supply of fermentable energy throughout the feeding cycle. Carbohydrates were dosed intraruminally at approximately 20% of total DMI twice daily before each feeding (Table 1Go, footnote "c" provides exact quantities of CHO dosed). Cows were fed at 0600 and 1800 at 95% of ad libitum intake determined at the beginning of the experiment. 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 consisted of 14 d for adaptation to the diet and 7 d for sampling.


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  		Table 1. Composition of the basal diet fed in the trial
 
Markers, Sampling, and Related Analyses
Ruminal ammonia N was labeled through a pulse-dose of 10 g/cow of 20 atom percent excess (APE) (15NH4)2SO4 (Cambridge Isotope Laboratories, Inc., Andover, MA) dissolved in 7 L of McDougall’s buffer (McDougall, 1948Go). The cows’ rumens were emptied in large carts on d 15 before the 0600 feeding, weighed, and the ruminal contents were thoroughly mixed by hand. A background ruminal sample was collected and the isotope, CHO, and Cr and Yb were added to the ruminal contents; a 0-h sample was collected following a thorough mixing, and the ruminal contents were returned to the cows’ rumens. Chromium-EDTA (equivalent of 2.5 g of Cr/cow; Udén et al., 1980Go) was used as a ruminal liquid passage rate marker. Ytterbium-labeled alfalfa hay (Hristov and Broderick, 1996Go), dosed at 300 g/cow (equivalent to 2.3 g of Yb), was used to estimate solid passage rates.

Ruminal Sampling.
Whole ruminal contents samples were collected at 0.5, 1, 2, 4, 6, 8, 10, 14, 18, 24, and 30 h following the dosing of markers at 0600 on d 15 of each period. Two additional samples, at 34 and 38 h, were collected for Yb analysis. Ruminal samples were collected from four locations in the rumen and the reticulum (approximately 250 g each), composited, and analyzed for DM, OM, and 15N enrichment of ammonia N and nonammonia N (NAN), and purine concentration in bacterial pellets (Hristov and Ropp, 2003Go). Purines were analyzed according to Zinn and Owens (1986)Go using the modified washing solution of Aharoni and Tagari (1991)Go and 0.6 M HClO4 (Makkar and Becker, 1999Go). Aliquots of the ruminal samples were filtered through cheesecloth, centrifuged (20,000 x g for 15 min at 4°C), and the supernatant fluid was analyzed for Cr. The solids were then analyzed for Yb concentration (Iris ICP atomic emission spectrophotometer; Thermo Jarrell Ash Corp., Franklin, MA). Fractional outflow rates of the ruminal fluid and solid phases were calculated as ln-transformed Cr or Yb concentrations plotted vs. time. Aliquots of the ruminal cheesecloth filtrates were immediately analyzed for pH and processed for analyses of ammonia, VFA, and reducing sugars (RS; Hristov et al., 1999Go), protozoal counts, and polysaccharide-degrading (carboxymethylcellulase, amylase, and xylanase) and deaminative activities (Hristov et al., 2001bGo). Individual ruminal samples were analyzed for ammonia and pH; the remaining analyses were performed on composite (volume base) samples. To determine rate of ruminal disappearance of supplemental dextrose and starch, aliquots of the whole ruminal contents samples collected between the 0600 and 1800 feedings (0.5, 1, 2, 4, 6, 8, and 10 h following marker and CHO dosing) from cows on GLU and STA treatments were freeze-dried and analyzed for RS or starch (starch analysis kit, Megazyme Int. Ireland, Ltd., Wicklow, Ireland; McCleary et al., 1994Go), respectively. Reducing sugars were extracted by agitating 1 g of dry sample with 20 mL of distilled water and 0.5 mL of 10% (wt/vol) sodium azide (Sigma Chemical Co., St. Louis, MO) for 30 min. Following centrifugation at 20,000 x g for 15 min at 4°C, supernatant fluid was analyzed for RS. Concentrations of RS and starch were plotted vs. time (h) after feeding/CHO dosing and curves fitted to a three-parameter, single exponential decay model (f = y0 + a x exp[–b x x]; Sigma Plot 8.0, SPSS Inc., Chicago, IL).

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 collected: one for analyses of milk fat, true protein and MUN and another for analysis of 15N-enrichment of milk protein (Hristov and Ropp, 2003Go).

Calculations.
The pool size of ruminal ammonia N was calculated as: ruminal weight (kg, from ruminal evacuation on d 15) x average ammonia N concentration (g/kg, from ruminal sampling). Ruminal ammonia (see Figure 3Go), bacteria (see Figure 4Go), and milk protein (see Figure 5Go) 15N-enrichment (APE) curves were plotted vs. time (h) and fitted to a three-parameter single exponential decay model (f = y0 + a x exp[–b x x]), a four-parameter, double-exponential decay model (f = y0 + a x exp[–b x x] + c x exp[–d x x]; Sigma Plot 8.0), or a double-exponential model of the type Y = a x exp(–c1 x x) x exp(–b x exp[–c2 x x]); (Dhanoa et al., 1985Go; PROC NLIN; SAS Inst., Inc., Cary, NC), respectively. Criteria for best fit were as previously described (Hristov and Ropp, 2003Go), or the proportion of the variance explained by the model in the case of milk protein. The average adjusted r2 for the ruminal ammonia N and bacterial N models were 0.98 ± 0.004 and 0.97 ± 0.006, respectively. The average proportion of the variance explained by the milk protein model (regression sum of squares/ uncorrected total sum of squares) was 0.99 ± 0.002. Areas under the predicted milk protein, ruminal ammonia, and bacterial 15N curves (AUC; 15N APE x 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, 1974Go).



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  		Figure 3. Effect of carbohydrate source on 15N-enrichment of ruminal ammonia N in dairy cows (means ± SE). GLU = corn dextrose; STA = cornstarch; NDF = white oat fiber, and MIX = (25% of each), apple pectin, GLU, STA, and NDF. Overall treatment effect, P = 0.11.

 


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  		Figure 4. Effect of carbohydrate source on 15N enrichment of ruminal bacterial N in dairy cows (means ± SE). GLU = corn dextrose; STA = cornstarch; NDF = white oat fiber, and MIX = (25% of each), apple pectin, GLU, STA, and NDF. Overall treatment effect, P = 0.48.

 


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  		Figure 5. Effect of carbohydrate source on 15N-enrichment of milk protein N in dairy cows (means ± SE). GLU = corn dextrose; STA = cornstarch; NDF = white oat fiber, and MIX = (25% of each), apple pectin, GLU, STA, and NDF. Overall treatment effect, P = 0.10.

 
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 the difference according to Nolan and Leng (1974)Go. The microbial N leaving the rumen that originated from ruminal ammonia N was calculated as follows: microbial N flow x 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 x proportion of bacterial N derived from ammonia N]/ irreversible loss of ammonia N) x 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, 2003Go) and by its 15N-enrichment. Data were presented as 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 x/(b + x); (see Figure 6Go; PROC NLIN, SAS). In this case, a represented the theoretical maximum of 15N secreted with milk protein as a percentage of 15N dosed in the rumen. The average proportion of the variance explained by the model (regression sum of squares/uncorrected total sum of squares) was 0.92 ± 0.024. Estimated maximum secretions and overall secretion lines were compared among treatments using dummy variable regression technique (Hristov and Ropp, 2003Go).



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  		Figure 6. Effect of carbohydrate source on cumulative secretion of 15N in milk protein (as percentage of 15N dosed intraruminally). Symbols are measured and lines are predicted values (single rectangular two-parameter hyperbola model). GLU = corn dextrose; STA = cornstarch; NDF = white oat fiber, and MIX = (25% of each), apple pectin, GLU, STA, and NDF. Significant differences between 15N secretion lines: STA vs. GLU, P = 0.01; STA vs. NDF, P = 0.001; MIX vs. NDF, P = 0.09.

 
Assuming corn dextrose (GLU treatment) was associated with the liquid and starch (STA treatment) with the solid phase of ruminal contents, the rates of digestion of the two CHO were calculated as: rate of digestion, h–1 = total rate of disappearance from the rumen, h–1 – rate of outflow of ruminal fluid (GLU) from Cr concentration data, or ruminal solids (STA) from Yb concentration data, h–1.

Nutrient Flow at the Duodenum and Total-Tract Digestibility Determinations
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)Go. Lithium/Co-EDTA and indigestible NDF (INDF) were used as liquid and solid phase markers, respectively. Total-tract apparent digestibility of DM, OM, N, and NDF were determined using acid-insoluble ash (AIA; Van Keulen and Young, 1977Go) as an internal digestibility marker. Intake of DM and nutrients and AIA and INDF concentration of the diet were corrected for CHO dosed intraruminally. Marker dosing, sampling, processing of samples, and analyses are given in Hristov and Ropp (2003)Go.

Other Analyses
The basal diet was sampled once each week. Samples were composited per period, dried at 60°C to constant weight (approximately 72 h) in a forced-air oven, and analyzed for ash (AOAC, 1999Go), AIA, N (Hristov et al., 2001aGo), and NDF (Ankom 200 fiber analyzer, Ankom Technology). A heat-stable amylase ({alpha}-amylase, EC No. 232-560-9; Sigma Chemical Co.) was used in the NDF analysis; sodium sulfite was not used in the analysis (Van Soest et al., 1991Go).

Total urine was collected 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, 2003Go).

On the last 2 d of each period, blood samples were collected from the jugular vein before (0 h) and 6 h after the morning feeding. Plasma was collected after centrifugation at 1,500 x g for 40 min, frozen at –40°C, and later analyzed for urea N (PUN; urea nitrogen kit, catalog No. 640-8; Sigma Diagnostics).

Statistical Analyses
All data were analyzed using SAS software. Intake, ruminal fermentation (except ammonia and pH), duodenal nutrient flows, digestibility, urine, and isotope data were analyzed by ANOVA Latin square (PROC MIXED). The model used was:


[1]

where µ is the overall mean, C, P, and T are 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). Cow was included as a random effect. Ruminal pH and ammonia data were analyzed as Latin square repeated measures with cow as a random effect. Statistical analysis of total protozoal counts was performed on log10-transformed data. Significant differences were declared at P ≤ 0.11. When the overall treatment effect was P ≤ 0.11, treatment means were separated by pairwise t-test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Implications
 Literature Cited
 
The target inclusion of CHO was 20% of total DMI. Due to variation in DMI with the basal diet, the average proportions of CHO dosed to the cows were as follows: GLU, 20.7%; STA, 20.8%; NDF, 20.0%; and MIX, 20.8% of DMI (Table 1Go).

The two CHO containing glucose, GLU and MIX, produced the lowest (throughout the course of sampling; P < 0.05) ruminal pH (Table 2Go). Average pH was also lower (P < 0.05) for STA compared with NDF. The GLU treatment, and to a lesser extent MIX, resulted in a noticeable decrease in pH immediately after CHO dosing (Figure 1Go); STA decreased pH in a similar pattern, but the lowest pH occurred between 2 and 4 h after the dose. There was an interaction (P < 0.001) between CHO source and time of sampling. Ruminal pH was lower (P < 0.001) for GLU compared with all other CHO at 0 (except MIX), 0.5, and 1 h and compared with NDF at 2 h after feeding/CHO dosing. Similarly, pH was lower (P < 0.001) for MIX compared with NDF at 0, 0.5, 1, and 2 h and compared with STA at 0, 0.5, and 1 h. Ruminal pH was lower (P = 0.06) for STA compared with NDF at 1 h, and was lower compared with MIX and NDF at 2 h (P = 0.01 and P < 0.001, respectively), and compared with all CHO at 4 h (P = 0.02 to P < 0.001). The MIX treatment had lower (P = 0.03 to P = 0.004) pH than all other CHO at 8 h after feeding. At 14 h (or 2 h after the 1800 feeding), pH of ruminal fluid was greater (P = 0.01 to P < 0.001) for NDF compared with all other CHO. The lowest overall ammonia concentration was associated with GLU and STA, MIX was intermediate, and NDF had the highest ammonia concentrations in the rumen (P < 0.05; there was no interaction between CHO and time of sampling for this variable; P = 0.33). Treatments GLU and STA maintained the lowest postfeeding ammonia concentrations (P = 0.02 to 0.002 vs. NDF and MIX; Figure 2Go). In accordance, NDF and MIX had larger ammonia N pools than GLU and STA (P < 0.05 compared with GLU, and P = 0.11 compared with STA). Concentration of RS was greater (P < 0.05) for GLU compared with STA, NDF, and MIX. The STA and MIX treatments had greater (P < 0.05) RS concentration than NDF. Protozoal counts were not affected by treatment (P = 0.20). The GLU treatment had the lowest total VFA concentration compared with all other CHO (P < 0.05), which was primarily due to lower acetate concentration (P < 0.05; Table 2Go). Propionate was not affected by treatment (P = 0.76), but butyrate concentration was greater for GLU, followed by STA, MIX, and NDF (P < 0.05). Concentrations of isobutyrate and isovalerate were dramatically decreased (P < 0.05) by GLU compared with the other CHO. Isobutyrate concentration also was decreased (P < 0.05) by STA and MIX compared with NDF. Isovalerate concentration was lowered (P < 0.05) by MIX compared with STA. Reflecting acetate concentration, acetate:propionate ratio was on average 19% lower (P < 0.05) for GLU compared with the other CHO. The carboxymethylcellulase activity of ruminal fluid was not affected (P = 0.23) by treatment. The NDF and MIX treatments resulted in greater (P < 0.05) xylanase activity of ruminal fluid than GLU. Amylase activity was greater (P ≤ 0.11) with the starch-containing CHO, STA and MIX, compared with GLU and NDF. Fractional outflow rates of ruminal fluid and solids were not affected (P = 0.62 and 0.37, respectively) by treatment. Before feeding, cows had similar (P = 0.25) amounts of microbial N (MN) in their ruminal contents, representing 50 to 60% of the total NAN in the rumen. Microbial N flow was decreased (P < 0.05) by NDF compared with the other CHO. The outflow of MN formed from ammonia N from the rumen was increased (P < 0.05) with STA compared with GLU and NDF. Expressed per kilogram of OM truly digested in the rumen, MN flow was decreased by NDF compared with the other CHO (P < 0.05). Urinary allantoin excretion did not seem to parallel the MN flow data and was not affected (P = 0.28) by treatment.


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  		Table 2. Effect of carbohydrate source on ruminal fermentation in dairy cows (n = 16)
 


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  		Figure 1. Effect of carbohydrate source on ruminal pH in dairy cows (means ± SE). GLU = corn dextrose; STA = cornstarch; NDF = white oat fiber, and MIX = (25% of each) apple pectin, GLU, STA, and NDF. Overall treatment effect, P < 0.001; treatment x time of sampling interaction, P < 0.001. Backrd = background.

 


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  		Figure 2. Effect of carbohydrate source on ruminal ammonia concentration in dairy cows (means ± SE). GLU = corn dextrose; STA = cornstarch; NDF = white oat fiber, and MIX = (25% of each), apple pectin, GLU, STA, and NDF. Overall treatment effect, P < 0.001; treatment x time of sampling interaction, P = 0.33. Backrd = background.

 
Intake of DM, OM, and N did not differ (P = 0.17, 0.46, and 0.22, respectively) among treatments (Table 3Go). Intake of NDF was greater (P < 0.05) for NDF compared with the other CHO and was greater (P < 0.05) with MIX compared with GLU and STA. Ruminal true digestibility of DM, OM, and N and digestibility of NDF were not different (P = 0.52, 0.64, 0.30, and 0.23, respectively) among treatments. Total-tract apparent digestibility of DM was decreased (P < 0.05) by GLU compared with NDF and was greater (P ≤ 0.11) for NDF and MIX compared with STA and GLU, respectively. Organic matter digestibility was not affected (P = 0.21) by treatment. The NDF and MIX treatments resulted in greater (P < 0.05) digestibility of NDF compared with STA and GLU, respectively, and MIX had greater (P ≤ 0.11) NDF digestibility than STA. Digestibility of dietary N was greater (P < 0.05) for NDF compared with the other CHO. The MIX treatment had greater N digestibility compared with GLU and STA (P < 0.05 and P ≤ 0.11, respectively). Fecal C:N ratio was increased (P < 0.05) by NDF compared with the other CHO; the ratio was greater (P < 0.05) for MIX compared with STA.


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  		Table 3. Effect of carbohydrate source on nutrient intakes and digestibility in dairy cows (n = 16)
 
Secretion of N with milk protein was not affected (P = 0.14) by treatment (Table 4Go). Milk N efficiency was greater for GLU compared with NDF and MIX (P < 0.05) and with STA (P ≤ 0.11). Absolute excretion of urinary N was decreased by GLU compared with NDF (P < 0.05) or MIX (P ≤ 0.11) and was lower (P < 0.05) for STA than for NDF. As a proportion of N intake, urinary N losses were similar among treatments (P = 0.30). Absolute and relative fecal N losses were the lowest (P < 0.05) for NDF compared with all other CHO. Relative fecal N losses were also decreased by MIX compared with GLU and STA (P < 0.05 and P ≤ 0.11, respectively). Total (urinary and fecal) N losses were not affected (P = 0.73) by CHO. There was no overall effect of treatment on concentration of PUN (P = 0.13). Concentration of MUN was decreased (P < 0.05) by GLU and STA compared with NDF and with MIX. The MIX treatment resulted in lower (P < 0.05) MUN concentration than NDF.


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  		Table 4. Effect of carbohydrate source on nitrogen losses and plasma (PUN) and milk (MUN) urea N concentrations in dairy cows (n = 16)
 
The 15N enrichment of ruminal ammonia N reflected ruminal ammonia concentrations and was greater for GLU compared with NDF (P < 0.05) and STA and MIX (P ≤ 0.11; Table 5Go). Average 15N enrichment of bacterial N was not different (P = 0.48) among CHO. The 15N ammonia and bacteria AUC were not affected (P = 0.14 and P = 0.51, respectively) by treatment. Enrichment of ruminal ammonia N rapidly decreased following the 15N dose and reached pre-dose levels within 5 to 6 h (Figure 3Go). The decrease in 15N enrichment of bacterial N was exponential and reached levels close to natural abundance within 30 h after the dose (Figure 4Go).


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  		Table 5. Effect of carbohydrate source on 15N enrichment of various N pools and 15N calculations in dairy cows (n = 16)
 
Based on AUC data, 38 to 60% of ruminal bacterial N originated from ammonia N, 45% of milk protein N originated from ruminal bacterial N (P = 0.65), and 17 to 28% of milk protein N originated from ruminal ammonia N. More (P < 0.05) bacterial N was formed from ammonia N with STA compared with GLU, MIX, and NDF, which resulted in a greater proportion of milk protein N derived from ammonia N for STA compared with GLU and MIX (P < 0.05) and with NDF (P ≤ 0.11). The proportion of bacterial N formed from ammonia N was greater (P ≤ 0.11) for NDF and MIX compared with GLU. A greater (P ≤ 0.11) proportion of milk protein N was formed from ruminal ammonia N with NDF than with GLU. Uptake of 15N-ammonia was very rapid and within the time required to process the 0-h sample, 8.4% (SE = 1.43; P = 0.84) of bacterial N was already formed from ammonia N. Within 30 min after the dose, 9 (GLU; P < 0.05 compared with NDF and MIX) to 14% (MIX; P < 0.05 compared with STA and GLU) of the bacterial protein was synthesized from ammonia N. More (P < 0.05) ammonia N was irreversibly lost from the ammonia N pool with STA and NDF compared with GLU. As a proportion of N intake, the irreversible loss of ammonia N was greater for STA than for GLU (P < 0.05) and for NDF than for GLU (P ≤ 0.11). Ruminal ammonia flux (absolute or as proportion of N intake) was decreased (P < 0.05) by GLU compared with the other CHO. Recycling of ammonia N was not affected (P = 0.21) by treatment. The efficiency of utilization of ruminal ammonia N for microbial protein synthesis ranged from 23 to 33% and was lower (P < 0.05) for NDF compared with the other CHO.

Cows were in late lactation consuming an energy-deficient diet, milk yield was low (18.3 ± 1.88 kg/d), and was not affected (P = 0.18) by treatment. Milk fat content averaged 3.41 ± 0.171% and was not affected (P = 0.86) by treatment. Milk true protein concentration was decreased (P < 0.05) by NDF compared with the other CHO (2.98 vs. 3.20%; SE = 0.12). Average 15N enrichment of milk protein N was greater for STA compared with NDF and MIX (P < 0.05) and with GLU (P ≤ 0.11). The milk protein 15N AUC was not affected (P = 0.14) by treatment. Secretion of the isotope in milk was bell-shaped (Figure 5Go). In all treatments, peak 15N concentration was measured at 15 h post-dose. The theoretical maximum of cumulative secretion of the isotope in milk protein N ranged from 8.8 to 10.3% of the ruminal 15N dose and was not affected by treatment (P = 0.97 to 0.48). Overall, cumulative 15N secretion in milk protein (Figure 6Go) was greater for STA compared with GLU and NDF (P = 0.01 and P = 0.001, respectively). Secretion of the isotope for MIX was also greater (P = 0.09) compared with NDF.

The concentration of RS in ruminal contents (GLU cows) rapidly decreased in an exponential manner after GLU dose (Figure 7Go) and the rate of total disappearance was greater (P < 0.05) than the rate of disappearance of STA (STA cows; Figure 8Go): 1.24 vs. 0.49/h, respectively. Assuming GLU was flowing out of the rumen with the fluid phase and STA with the solid phase, average rates of digestion for these two CHO were calculated to be 1.15 and 0.46/h, respectively.



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  		Figure 7. Decrease in reducing sugars concentration in the rumen of corn dextrose (GLU)-treated cows with time after GLU dose (means ± SE).

 


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  		Figure 8. Decrease in starch concentration in the rumen of cornstarch (STA)-treated cows with time after STA dose (means ± SE).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Implications
 Literature Cited
 
Earlier studies investigating the effects of CHO supplementation and type on ruminal fermentation focused on the possibility of using nonprotein N (urea) as a substitute for feed protein in cattle diets (Mills et al., 1944Go; Belasco, 1956Go). More recently, the target of CHO supplementation was improving dietary CP and ammonia utilization through manipulation of ruminal microbial fermentation (Chamberlain et al., 1985Go; Rooke et al., 1987Go; Oh et al., 1999Go).

In the present study, the effect of the rapidly fermentable CHO on ruminal fermentation was noticeable immediately after dosing. At 0.5 to 1 h, average ruminal pH was 5.3 for GLU, which was 10% less than the average pH of MIX (5.9) at these sampling times. The lowest pH for STA (5.6) was similar to that for GLU, but occurred between 2 and 4 h after CHO dosing. Reduction of ruminal pH in dairy cows as a result of sugar supplementation was observed by Kim et al. (1999a)Go, but not by McCormick et al. (2001)Go or Osborne et al. (2002)Go. Ammonia concentration was persistently lower for GLU and STA and slightly more variable for MIX compared with NDF. Apparently, provision of large amounts of GLU or STA had resulted in lowered ammonia concentration compared with the NDF treatment; however, it is possible that the mechanisms of ammonia reduction were different between GLU and STA. Although, both treatments resulted in a significant decrease in ruminal ammonia concentration and had a similar total microbial N flow to the small intestine, a significantly larger proportion of microbial protein was synthesized from ammonia N with STA than with GLU, which resulted in a greater flow of microbial N synthesized from ammonia. Thus, the apparently more rapid degradation of GLU provided an immediate source of energy to the ruminal microorganisms and feed AA N was most likely rapidly utilized without passing through the ammonia N pool. With STA, a larger proportion of alfalfa protein/AA N was broken down and deaminated, but eventually incorporated as ammonia N by the ruminal microorganisms. The decreased concentrations of isobutyrate and isovalerate and decreased irreversible ammonia N loss and flux with GLU compared with STA support this hypothesis. In the rumen, the branched-chain VFA, are derived from branched-chain AA (Wolin et al., 1997Go). Our results most likely reflect decreased branched-chain AA concentration in the rumen with GLU compared with the other CHO due to enhanced microbial uptake or reduced production. Decreased ammonia concentration in the rumen has been commonly reported in vitro when CHO are added to the incubation media (Russell et al., 1983Go; Hristov et al., 1997Go; Lee et al., 2003Go). Similarly, ammonia concentration was decreased in vivo with the addition of glucose, starch (Chamberlain et al., 1985Go; Heldt et al., 1999Go; Osborne et al., 2002Go), sucrose, xylose (Huhtanen, 1987Go; Khalili and Huhtanen, 1991Go), or maltodextrin (Kim et al., 1999aGo), although in some cases, sugar addition had no effect (McCormick et al., 2001Go; Sannes et al., 2002Go). In vivo studies directly comparing sugar to starch reported lower ammonia concentration with the former CHO (Chamberlain et al., 1993Go; Oh et al., 1999Go). In the current study, the decreased ammonia levels with MIX (compared with NDF) are due to the corn dextrose and starch component of this treatment. The GLU treatment drastically decreased acetate and consequently concentration of total VFA in the rumen compared with the other CHO. This decrease does not correspond well with the low pH with GLU. Although, lactate was not measured in this study, it seems likely that lactate production and concentration in the rumen may have been increased by GLU. Many ruminal bacteria, particularly fibrolytic species, cannot tolerate low pH (Russell et al., 1992Go) and the observed reduction in acetate concentration may be a result of decreased activity of structural carbohydrate digesters. Some of the polysaccharide-degrading activities, particularly xylanase activity, were also decreased by GLU. More efficient use of ATP (Strobel and Russell, 1986Go; Russell, 1992Go) with GLU can also be partially responsible for the decreased VFA with this treatment. The increased butyrate concentration with GLU is probably indicative of stimulated growth of the major butyrate producer in the rumen, Butyrivibrio fibrisolvens. None of these effects were related to changes in protozoal counts. Effects of CHO on VFA concentration and ratios reported in the literature are variable. In a design similar to the present experiment, but with sheep, Chamberlain et al. (1985)Go found only numerical reduction in the proportion of acetate and numerical increases in the proportions of propionate and butyrate with sugar (glucose or sucrose) supplementation at 13% of DMI. In another experiment from the same study, the authors reported a significant reduction of the molar proportion of acetate and increased proportion of propionate and butyrate with sucrose supplementation. Similar effects of sugar supplementation on acetate and butyrate concentrations in vivo or in vitro were reported by others (Huhtanen, 1987Go; Khalili and Huhtanen, 1991Go; Lee et al., 2003Go), but in many experiments shifts in VFA proportions were not observed (McCormick et al., 2001Go; Osborne et al., 2002Go; Sannes et al., 2002Go).

There were no differences among CHO in their effect on microbial N outflow from the rumen (except the negative control, NDF, which resulted in a 23% reduction in MN flow), which suggests a similar overall effect of the different rates of energy release on microbial protein synthesis in the rumen. The flow of bacterial N formed from ruminal ammonia N, however, was greater (by 33%) for STA than for GLU, which, as discussed earlier, was probably due to effects on energy availability immediately after CHO dosing and deamination/ uptake of alfalfa AA. In most published research, microbial N flow to the small intestine has been increased with addition of various sugars to the diet (Rooke et al., 1987Go; Khalili and Huhtanen, 1991Go; Kim et al., 1999bGo). In one study (Sannes et al., 2002Go), MN flow was decreased by sucrose added at 3.2% of dietary DM, but MN flow was determined indirectly (through urinary purine derivative excretion) and the authors had no clear explanation of the observed effect. Microbial protein synthesis in the rumen is primarily a function of availability of energy and N. The basal diet contained sufficient amounts of ruminally soluble and available N (from alfalfa hay), but was deficient in available energy (27% nonfiber CHO; NRC, 2001Go), and it is not surprising that addition of GLU, STA, or a combination of ruminally available CHO enhanced microbial protein synthesis and microbial N outflow from the rumen.

Through negative associative effects (Firkins, 1997Go), fiber digestion was decreased by sugar supplementation of the diet (Huhtanen, 1987Go; Heldt et al., 1999Go; Lee et al., 2003Go), but in some cases nonstructural CHO supplementation did not affect (Huhtanen and Robertson, 1988Go), or slightly increased (Rooke et al., 1987Go) fiber digestion. Although, ruminal digestibility was not affected by CHO supplementation in the present study, total tract apparent digestibility of DM, NDF, and N was clearly decreased by GLU or STA compared with the NDF treatment. Particularly interesting is the greater N digestibility with the NDF diet. Analysis of the data showed a lower N concentration in fecal DM of the cows subjected to the NDF treatment (1.8 vs. 2.6%, NDF vs. all other treatments). Carbon concentration was similar among treatments (44.5 vs. 44.4%). In addition to the numerically greater ruminal N degradability with NDF, a possible explanation for these results is decreased fermentation and microbial protein synthesis in the large intestine with NDF compared with the other CHO. Indeed, analysis of fecal matter for purines showed a greater (P = 0.06) concentration of total purines with STA than with NDF and GLU (6.0 vs. 4.5 and 5.5 mg/g, SE = 0.34). Thus, the significantly greater C:N ratio in feces from NDF-treated cows was a direct result of the decreased excretion of N with this diet.

Overall, due to the nature of the basal diet and the lactation stage of the cows, the efficiency of conversion of dietary N into milk protein N was low in this experiment. The increased milk N efficiency with GLU was a result of the numerically greater milk N yield and slightly lower N intake with this treatment compared with the other CHO. Inclusion of sugars in the diet of lactating dairy cows has not produced any significant effects on milk N efficiency (McCormick et al., 2001Go; Ordway et al., 2002Go; Sannes et al., 2002Go). There was a noticeable decrease in urinary N excretion with GLU and STA compared with NDF in the present experiment; however, considerably less N was excreted in feces with the NDF diet, the reason for which was discussed earlier. Thus, overall N losses were not different among CHO. There is an important difference between N lost with urine and N lost with feces. The main form of N in urine, which can represent up to 77% of the total N excreted by dairy cows, is urea (Bristow et al., 1992Go); urinary N excretions can vary from 80 to 320 g/d (Whitehead, 1995Go) and ammonia emitted from livestock manure is mainly a product of urinary urea breakdown (Rom and Dahl, 1997Go). If mixed with feces, urea is quickly converted into ammonia by the abundant urease activity present in fecal matter. Depending on factors such as pH, temperature, and concentration, a large proportion of ammonia can be rapidly volatilized and lost to the environment. In contrast, fecal N is more stable and contributes little to N emissions from manure (Satter et al., 2002Go). Thus, from an environmental point of view, the diets providing ruminally available CHO (GLU and STA), would potentially result in a lower rate of manure N volatilization compared with the NDF-containing diets. The decreased MUN (and PUN) with the GLU and STA diets reflected increased capture of ammonia N in the rumen and seemed to be related to the amount of ruminally fermentable energy introduced to the rumen with CHO. Huhtanen and Robertson (1988)Go and Sannes et al. (2002Go; urinary urea) also observed decreased urinary N losses with sugar supplementation. Plasma urea N concentration was decreased with maltodexrin (Kim et al., 1999aGo), or glucose (Osborne et al., 2002Go) supplementation of the diet of dairy cows, but no effects were reported by Sannes et al. (2002)Go or Ordway et al. (2002)Go. Others have observed increased PUN (McCormick et al., 2001Go) with sucrose supplementation. Milk urea N levels were either decreased (Sannes et al., 2002Go), remained unchanged (McCormick et al., 2001Go; Ordway et al., 2002Go), or were increased (Cherney et al., 2003Go) by sugar supplementation of dairy cow diets. The data of Cherney et al. (2003)Go deserve further attention. These authors explained the lack of effect of sucrose supplementation on ruminal fermentation and consequently urinary N excretion, which was similar between the supplemented and un-supplemented diets, and the increase in MUN concentration by the high outflow rate of sucrose from the rumen preventing its utilization by ruminal bacteria as an energy source. Results from the present experiment, however, suggested a very high digestion rate of GLU compared with a relatively slow rate of ruminal fluid passage, clearly indicating that soluble sugars would be largely digested by the ruminal microbiota. The different responses in PUN or MUN between our experiment and those of McCormick et al. (2001)Go, Ordway et al. (2002)Go, and Cherney et al. (2003)Go can be attributed to the different composition of the basal diets (energy-deficient, all-forage diet vs. balanced, forage-concentrate diets) and associative effects among feed ingredients.

Due to the lower ruminal ammonia N concentration and smaller pool size, average 15N enrichment of ammonia N for GLU was greater compared with some of the other CHO. Bacterial N formed from ammonia N ranged from 38 to 60%. The main difference in the proportion of bacterial N synthesized from ammonia N was between GLU and STA. As discussed earlier, the more rapid release of ruminally fermentable energy with GLU, compared with STA, led to decreased breakdown of alfalfa AA and greater uptake of preformed AA rather than ammonia N by the ruminal microorganisms. The more active uptake of ammonia with STA than with NDF (or MIX) was a result of the greater availability of ruminally fermentable energy with the former treatment. The reason why ammonia uptake was not decreased to a greater extent by NDF can be found in the documented preference of ruminal cellulolytic bacteria for ammonia N (Russell et al., 1992Go). The daily amount of ammonia N leaving and not returning to the ruminal ammonia N pool (i.e., the irreversible loss of N from this pool) would represent ammonia leaving the rumen as microbial N, absorbed through the ruminal wall, and ammonia outflow with digesta. Due to the decreased uptake of ammonia N by the ruminal microorganisms, the irreversible loss was significantly lower for GLU than for STA (or NDF). Similarly, the ammonia N flux through the ruminal ammonia pool was significantly lower for GLU compared with all other CHO, reflecting decreased ammonia production with the former CHO. The differences between STA and the NDF-containing treatments did not reach significance, but also suggest a numerically decreased flux of N through the ammonia N pool with STA. Although non-significant and variable between cows, ammonia N recycling data paralleled the trends in ammonia flux and irreversible loss. Siddons et al. (1985)Go reported 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 reduction in ruminal ammonia is due to absorption. Our data indicate that between 23 and 33% of the irreversible ammonia N loss was with the microbial N leaving the rumen and 77 to 67% was due to absorption or outflow with the ruminal fluid phase, respectively. Given that estimated proportions of milk protein N originating from bacterial N were similar among treatments, the proportions of milk protein N formed from ruminal ammonia N paralleled the proportion of bacterial N synthesized from ammonia N data. On average, 55% of milk proteins were synthesized from nonbacterial N sources. The average NAN flow to the duodenum varied from 428 (NDF) to 536 (GLU) g/d, of which microbial N flow comprised 153 and 197 g/d, respectively. If NRC (2001)Go efficiency coefficients are used (0.64 and 0.80 for microbial and ruminally undegraded [RUP] N, respectively), these flows represent 98 and 126 g/d of metabolizable protein N from microbial and 220 and 276 g/d from nonmicrobial sources (NDF and GLU, respectively). The isotope data (Table 5Go) indicate 42 and 43 g/d of milk protein N output from microbial and 48 and 54 g/d from RUP sources for the two diets (NDF and GLU), respectively. Thus, 43 and 34% of the estimated MP flow from microbial protein and 22 and 20% from RUP (NDF and GLU, 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, 1974Go). Therefore, the above coefficients may represent the demand for AA by the mammary gland, with respect to the stage of lactation and level of production of the experimental cows, but could also suggest differences in utilization of metabolizable protein from microbial and RUP sources. Raggio et al. (2003)Go demonstrated that the efficiency of utilization of certain AA for milk protein synthesis decreased linearly with increasing metabolizable protein supply. Some authors suggested lower efficiency of utilization of microbial amino acids (due to imbalanced AA profile) compared with casein (Cant et al., 1999Go).

Overall, more ruminal ammonia 15N was recovered in milk protein with STA than with NDF or GLU, suggesting increased efficiency of utilization of ruminal ammonia N for milk protein synthesis with the former CHO. Our approach in estimating the proportion of 15N ammonia used for milk protein synthesis was based on the assumption that most of the ruminal ammonia that is not utilized 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., 1995Go), and 80 to 90% of the 15N infused appeared in urea (Lobley et al., 1996Go). 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 4Go). Milk protein 15N enrichment also decreased rapidly within 100 h after dosing of the isotope (or approximately 70 h after the decline in bacterial enrichment; Figure 5Go). 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. Apparently, 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 present study (as a proportion of 15N infused in the rumen) was similar to our previous estimates (12 to 14%: Hristov and Ropp, 2003Go; 9%: Hristov et al., 2004Go). Recalculation of the data by Petri et al. (1988)Go shows that in the lactating goat, from 10 to 14% of the irreversible loss of ruminal ammonia N was recovered in milk protein via microbial protein.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
Literature Cited
 
This experiment demonstrated that provision of readily fermentable energy can decrease ammonia concentrations in the rumen through inhibited production of ammonia and enhanced incorporation of pre-formed feed amino acids, or through enhanced uptake of ammonia for microbial protein synthesis. Decreased production or enhanced utilization of ammonia in the rumen resulted in a shift in nitrogen excretion from urine to feces. Energy that is rapidly fermentable in the rumen would decrease ammonia production and flux, and may decrease ammonia nitrogen cycling, but the overall efficiency of ammonia utilization for milk protein synthesis can only be increased by enhancing ruminal microbial ammonia uptake.


   Footnotes
 
1 This study was partially supported by funds from the United Dairymen of Idaho, the Idaho Agric. Exp. Stn., and a USDA/NRI grant (No. 0103316). The authors would like to thank W. Price for assistance with statistical evaluation of the results, and the staff of the Dept. of Anim. and Vet. Sci. Experimental Dairy for their conscientious care of the experimental cows. Back

2 Correspondence: P.O. Box 442330 (phone: 208-885-7204; fax: 208-885-6420; e-mail: ahristov{at}uidaho.edu).

Received for publication June 28, 2004. Accepted for publication November 19, 2004.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Implications
 Literature Cited
 


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