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MTT Agrifood Research Finland, FIN-31600 Jokioinen, Finland
2 Correspondence:
phone: +358 3 4188 3662; fax: +358 3 4188 3661; E-mail:
seppo.ahvenjarvi{at}mtt.fi.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Dairy Cows • Rumen Bacteria • Rumen Metabolism • Rumen Protozoa
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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The objective of the present study was to quantify the flow of microbial and nonmicrobial N leaving the rumen of dairy cows fed forage diets supplemented with energy and protein rich concentrates. To minimize the contribution of endogenous N to nonmicrobial N flow, digesta samples were obtained from the omasal canal (Ørskov et al., 1986). To account for differences in 15N enrichment between various microbial pools (Broderick and Merchen, 1992), the contribution of liquid- and particle-associated bacteria (LAB and PAB, respectively) and protozoa to nonammonia N (NAN) flow entering the omasal canal was also determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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). Cows averaged 571 kg of BW during the study. Silage was prepared from secondary growths of timothy grass (Phleum pratense) and red clover (Trifolium pratense) swards. Once cut, herbage was wilted and harvested using a precision chopper and ensiled using a formic acid-based additive (AIV-2; Kemira-Agro, Helsinki, Finland) applied at a rate of 5 L/t. Prior to feeding, barley was coarsely milled using a roller mill. Solvent-extracted rapeseed meal was obtained from a commercial source (Rehuraisio Ltd., Raisio, Finland).
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Sampling Procedures
Each experimental period lasted for 21 d. During the adaptation period (d 1 to 10), cows had free access to silage. Thereafter (d 11 to 21), silage offered was restricted to 95% of that during the adaptation period. Dry matter intake measured between d 17 to 20 are reported. Cows were fed twice daily at 0600 and 1800 and milked twice daily at 0700 and 1700. Milk yield was recorded daily, but only data from days 12 to 20 are reported. Milk samples were collected over four consecutive milkings (d 12 to 14) for the determination of milk protein, fat, and lactose.
Rumen fermentation characteristics were assessed in samples of rumen fluid collected on d 14 at 0600, 0800, 1000, 1200, 1400, and 1600. From d 15 at 1800 until the end of the sampling period, Yb-acetate (1.5 g/d of Yb) and LiCoEDTA (1.7 g/d of Co) were dissolved in 6 L distilled water and continuously infused into the rumen via separate lines following a priming dose of Yb-acetate (2.2 g of Yb) and LiCoEDTA (2.5 g of Co) into the rumen via the rumen cannula. From d 16 at 0600 until the end of the period, 10.9 g/d of ammonium sulfate (Isotec Inc., Miamisburg, OH) with a 10% enrichment of 15N (230 mg/d of 15N) was added to the LiCoEDTA infusate to enrich microbial N with 15N.
To determine digesta flow entering the omasal canal, spot samples (500 mL) of omasal canal digesta were collected into an insulated pre-warmed flask on d 18 at 1000, 1300, and 1600; on d 19 at 0830, 1430, and 1730; and on d 20 at 0700 and 1130 according to Huhtanen et al. (1997) and incorporating the modifications of Ahvenjärvi et al. (2001). To quantify protozoal N flow escaping the rumen, 700 mL of digesta was collected from the omasal canal into a pre-warmed insulated flask on d 18 at 0600, on d 19 at 1000, and on d 20 at 1400. To isolate particle- and liquid-associated bacteria, 1000 mL of reticular digesta was collected on d 20 at 0600, 1000, and 1400. A wide-necked 500-mL bottle, plugged with a rubber stopper, was placed into the ventral reticulum, and the stopper was removed. Once the bottle was filled with digesta, it was removed from the reticulorumen, and the contents were emptied into a 1000-mL bottle maintained at 39°C in a water bath. This procedure was repeated to yield 1,000 mL of reticular digesta. Spot samples of feces (500 mL) were obtained at the same time as omasal digesta samples were collected. On d 18 to 20, a total urine collection was performed using a light harness that was attached with adhesive around the vulva. Urine was preserved using 800 mL of 10-N H2SO4 added to the collection vessel on each collection day.
Preparation of Digesta Samples
Samples of rumen fluid were immediately subjected to measurements of pH and then prepared for the determination of VFA and ammonia according to Ahvenjärvi et al. (1999). Samples were stored at -20°C prior to analysis. Immediately after collection, samples of omasal canal digesta were filtered through one layer of cheesecloth, and the filtrand was washed with 200 mL of 39°C 0.9% (wt/vol) NaCl solution to remove loosely attached bacteria and protozoa into the filtrate. Filtrand, defined as large particles, was frozen at -20°C, pooled within animal, and subsequently lyophilized. Filtrate was frozen and stored at -20°C until the end of each period. At the end of each period, samples of filtrate were thawed, pooled within animal, and centrifuged at 10,000 x g for 30 min. The supernate, defined as liquid, was removed by aspiration. The pellet, defined as small particles, and a subsample of liquid were lyophilized. Lyophilized samples were transferred into a 50°C oven overnight to achieve a constant DM concentration before the weight of large particle, small particle, and liquid samples were recorded.
Immediately after collection, digesta samples used for the determination of protozoal N flow were filtered through two layers of cheesecloth into a flask incubated in a 39°C water bath. To displace all protozoa into the filtrate, filtrand was washed twice with 250 mL of 39°C buffer. Buffer was prepared according to Goering and Van Soest (1970) with the exception that trypticase was omitted and 1 g/L glucose was included. Filtrate was transferred into a 1000-mL volumetric cylinder and incubated for 30 min at 39°C. Flocculent material floating on the top was removed by aspiration, and volume was recorded. Measurements were corrected for the addition of buffer (500 mL less that retained by the cheesecloth) to calculate the volume of omasal canal liquid used to isolate protozoa. To sediment protozoa, samples were centrifuged at 1,000 x g for 10 min at 4°C using a swinging-out rotor. Supernate was removed by aspiration and discarded. Microscopical examination indicated that the protozoa-containing pellet was contaminated with fine feed particulate matter. Therefore, the pellet was washed with 500 mL of 0.9% NaCl solution on a polyester filter (17-µm pore size, 10% open area; Swiss Silk Bolting Cloth Mfg. Co. Ltd., Zurich, Switzerland). The 17-µm pore size was the smallest that allowed proper passage of feed particulate matter. Yellowish material remaining on the polyester filter was rinsed through a 200-µm polyester filter into a pre-weighed flask using distilled water, frozen at -20°C, and lyophilized. Lyophilized samples were kept in an oven at 50°C overnight to achieve a constant DM concentration before sample weight was recorded.
Digesta samples collected for the isolation of LAB and PAB were immediately filtered through four layers of cheesecloth into a flask incubated in ice slurry. Filtrand was washed twice with 250 mL of 39°C 0.9% (wt/vol) NaCl solution to dislodge loosely attached bacteria into the filtrate. Filtrate was centrifuged at 1,000 x g for 10 min at 4°C using a swinging-out rotor to sediment protozoa and feed particles. Supernate was removed by aspiration and centrifuged at 10,000 x g for 30 min at 4°C using a fixed-angle rotor. Supernatant was removed by aspiration and the pellet, defined as LAB, was lyophilized prior to chemical analysis. To detach bacteria firmly adherent to particulate matter, the following procedure was modified from that described by Minato and Suto (1978). Filtrand was incubated in an oven at 39°C for 15 min suspended in 800 mL of 0.9% (wt/vol) NaCl solution containing 1 g/L of carboxymethylcellulose (Sigma, St. Louis, MO). Thereafter, the sample was maintained at 4°C overnight. The next day, PAB were harvested using the same differential centrifugation procedure used for LAB.
Chemical Analysis
Dry matter of feed ingredients and feces was determined after 18 h in a forced-air oven at 105°C. Silage DM content was corrected for volatile losses according to Huida et al. (1986). For chemical analysis, feed ingredients and feces were dried to a constant weight at 60°C in a forced-air oven, and ground through a 1-mm screen. The DM concentration of air-equilibrated samples was determined after a 16-h incubation at 105°C. Digesta and feces were analyzed for Co and Yb according to Williams et al. (1962). To determine indigestible NDF concentration in feed ingredients, feces, large particles, and small particles, between 2 to 4 g of material were incubated in duplicate in polyester bags of 6-µm pore size for 12 d in the rumen of two cows according to Ahvenjärvi et al. (2000). The concentration of AIA in feed ingredients and feces was determined as described by Anon (1971). The concentration of ash was determined after ignition in a muffle furnace at 600°C for 18 h. Nitrogen concentration in fresh samples of silage, feces, and urine was determined by the Kjeldahl method using CuSO4 as a catalyst. Nitrogen concentration in other feed ingredients, digesta, and microbial samples was determined using a Dumas-type N analyzer (Leco FP-428; Leco Corporation, St. Joseph, MI). The VFA concentration of silage, ruminal fluid, and lyophilized liquid phase of omasal canal digesta (after resuspension in distilled water) was determined according to Huhtanen et al. (1998). Feed ingredients were analyzed for ether extract according to official method 920.39 (AOAC, 1990). The NDF concentration in feed ingredients, feces, and large particles was determined in the presence of Na2SO3 according to Van Soest et al. (1991). The ADF concentration of large particles was determined according to Robertson and Van Soest (1981). Because filtering problems were encountered in NDF analysis of small particles using crucibles, the NDF and ADF concentration in small particles was determined using an ANKOM 220 FiberAnalyzer (ANKOM Technology, Fairport, NY). Despite repeatable analysis of NDF for small particles, the NDF to ADF ratio in small particles was greater than that in large particles (5.7 vs 1.8). Therefore, the NDF concentration of small particles was calculated assuming the same NDF to ADF ratio as observed for large particles. To reduce the number of samples for analysis of 15N and ammonia N, small particles and liquid were combined to provide a composite sample according to their respective proportions in true digesta, calculated using the triple marker method described below. For 15N analysis, large particles, composite samples containing small particles and liquid, and microbial samples equal to 100 µg of N were weighed into tin capsules (PDZ Europa, Cheshire, UK) and 50 µL of KCO3 solution (10 g/L) were pipetted onto each sample. Samples were dried at 60°C overnight to remove ammonia residues. Enrichment of 15N was analyzed in triplicate using a Roboprep-CN analyzer (Europa Sientific Ltd., Crewe, UK) linked to a VG Micromass 622 mass spectrometer (VG Micromass Ltd., Winsford, UK) according to Esala (1991). The ammonia N concentration in large particles and in reconstituted composite samples of small particles and liquid was measured according to McCullough (1967). Milk samples were analyzed for protein, fat, and lactose using an infrared milk analyzer (Milkoscan; Foss Electric, Hillerod, Denmark). Concentrations of urea in milk were determined by difference following urease (Sigma) hydrolysis to ammonia.
Calculations
Daily fecal excretion was determined on the basis of indigestible NDF and AIA concentration in feed ingredients and feces. Because both markers indicated similar main treatment effects, the mean of these values has been reported. The chemical composition of omasal canal true digesta and the DM flow entering the omasal canal were calculated based on a triple marker method (France and Siddons, 1986) utilizing three indigestible markers: indigestible NDF, Yb, and Co. The flow of fresh matter in the liquid phase was corrected for added saline.
Because VFA entering the omasal canal can be considered as end products of rumen fermentation and, therefore, being digested in the rumen, the amount of VFA entering the omasal canal in liquid phase was subtracted from total OM flow. On average, the amount of VFA entering the omasal canal was 1.4 kg/d. Nonammonia N was calculated by difference between total N and ammonia N. Total NAN flow entering the omasal canal was divided into five fractions: particle-associated bacteria N (NPAB), liquid-associated bacteria N (NLAB), protozoal N, nonmicrobial N in large particles, and nonmicrobial N in small particles and liquid. Nonmicrobial N fractions contained dietary and endogenous N. In calculations, 15N-atom percentage excess is defined as 15N-atom% above background 15N-atom% measured in unlabelled dietary N. Background 15N-atom% averaged 0.3667. An alternative approach would have been to calculate 15N-atom percentage excess in each fraction (NPAB, NLAB, protozoal N, large particulate matter, and the composite phase of small particles and liquid) as a difference in 15N-atom% between labelled and unlabelled sample. Previous studies in our lab have indicated similar background 15N-atom% levels in microbes and digesta.
Particle-associated bacteria and protozoal N flow entering the omasal canal was calculated as
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It has been assumed that after the saline wash of large particulate matter, protozoa were entirely associated with the small particle and liquid composite sample. Justification for this approach is provided by the observations that following saline washing of ruminal particulate matter only 1 to 2% of protozoa remain attached (Coleman, 1985). Liquid-associated bacteria N flow was calculated as
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Apparent ruminal N degradability was calculated as [(N intake - NAN flow entering the omasal canal)/N intake]. True ruminal N degradability was calculated in two ways, either assuming that no endogenous N entered the omasal canal [(N intake - NAN flow - microbial N flow)/N intake] or assuming an endogenous N flow of 85 mg/kg BW0.75 (Ørskov et al. 1986) [(N intake - NAN flow - microbial N flow - endogenous N flow)/N intake]. Daily N retention was calculated as [N retention = N intake - (N in milk + N in feces + N in urine)].
Statistical Analysis
The effect of supplementation of diets with barley and rapeseed meal on feed intake, digesta flow, diet digestibility, ruminal fermentation, and microbial composition was assessed using the following statistical model with the MIXED procedure of SAS (Littell et al., 1998):
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where A is a random effect of animal, and P, B, and R are the fixed effects of period, barley, and rapeseed meal, respectively. Differences in chemical composition between rumen microbial pools (liquid- and particle-associated bacteria, and protozoa) and the effect of experimental diets on diurnal variation in rumen fermentation were assessed by the following model for repeated measures with the MIXED procedure of SAS:
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where M is a fixed effect of microbial pool (or effect of time after morning feeding), animal, main effect error (eijkl), and interactions between microbial pool (or time) and animal are random effects. Error term (eijklm) was used for the F test for microbial pool (or time) effect. Degrees of freedom were determined using the Satterthwaite option. Various covariance matrix structures (Compound symmetric, Autoregressive of order 1, and Toeplitz) were used, and the structure with the best fit was selected on the basis of Akaike’s information criterion. The best fit was achieved using either Compound symmetric or Autoregressive of order 1 matrix structures. Pairwise differences in chemical composition between microbial pools were determined using t-tests.
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Results |
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). Supplementation of grass-red clover silage with barley and rapeseed meal increased total DMI, but the effects were smaller when supplements were offered together than alone (Table 1). The amount of refusals was not affected by diet and averaged 0.91 kg/d DM during collection periods. Supplementation of diets with barley increased true OM degradability in the rumen and apparent OM digestibility in the total tract (Table 2). Supplementation of diets with rapeseed meal had no effect on OM degradability in the rumen or digestibility in the total tract. Both barley and rapeseed meal supplementation decreased NDF digestibility in the rumen and total tract.
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). Barley had no effect on rumen ammonia concentration, whereas, rapeseed meal resulted in a significant increase. Barley supplementation decreased the molar proportion of acetate, propionate, and isobutyrate, and increased that of butyrate and caproate. Supplementation with rapeseed meal decreased the molar proportion of acetate and increased that of valerate.
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). Despite large variations between diets in 15N enrichment, differences between rumen microbial pools were consistent for all diets, such that enrichment was highest in LAB and lowest in protozoa.
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). Supplementation of diets with rapeseed meal increased total NAN flow, nonmicrobial N flow in large particles, and total nonmicrobial N flow entering the omasal canal. Significant interactions between barley and rapeseed meal for fecal N excretion indicated that both barley and rapeseed meal increased N losses in feces, but the effect was more pronounced when supplements were fed separately than together. Interactions between barley and rapeseed meal for urinary N excretion (P = 0.05) indicated that rapeseed meal increased N lossess in urine, with the effect being greater when rapeseed meal was fed alone than together with barley. Barley and rapeseed meal supplements increased calculated daily N retention, with the effects being additive.
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). In contrast, rapeseed meal increased apparent ruminal N degradability but had no effect on true ruminal N degradability. A tendency (P = 0.06) for interactions between barley and rapeseed meal in total tract N digestibility indicated that barley and rapeseed meal increased N digestibility in the total tract, the effect being greater when the ingredients were fed separately than together. Barley and rapeseed meal had no effect on the efficiency of microbial N synthesis in the rumen. Supplementation of silage with barley and rapeseed meal increased the utilization of N for milk protein synthesis and N retention in body tissues, with the effects being additive.
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). Supplementation had no effect on milk fat concentration, whereas, barley increased that of milk protein. A significant interaction between barley and rapeseed meal for lactose indicated that rapeseed meal decreased lactose concentration when fed alone, but the effect was reduced when both barley and rapeseed meal were fed. Supplementation of diets with rapeseed meal tended to increase (P = 0.07) milk urea concentration. Barley and rapeseed meal increased milk fat, protein, and lactose yields. Milk yield efficiency, calculated as fat-corrected milk yield per kg digestible OM intake, indicated an interaction (P < 0.01) between barley and rapeseed meal. Barley decreased milk yield efficiency, but the effect was alleviated when barley and rapeseed meal supplements were fed together.
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Discussion |
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Consistent for all diets, virtually all NDF digestion occurred in the rumen. This finding could be explained by relatively low DMI (Table 1) which probably resulted in low ruminal passage rate and, therefore, high ruminal NDF digestibility and low potential digestibility of NDF flowing out of the rumen (Tamminga, 1993). Supplementation of diets with barley decreased ruminal and total tract digestibility of NDF by 3 and 5 percentage units, respectively (Table 2). Consistent with this observation, ruminal pH in barley-supplemented diets remained below 6.1 for extended periods (4 and 6 h for diets SB and SBR, respectively; data not presented). Levels of pH between 6.0 and 6.1 were suggested to be critical for fiber digestion (Mould et al., 1983). In heifers, a similar decrease in ruminal NDF digestibility (81 to 76 percentage units) was observed when grass silage was partly replaced by a barley based concentrate (Aronen and Vanhatalo, 1992). Decreases in fiber digestion, as a result of supplementation of forage diets with readily fermentable carbohydrates, have been extensively reported (Hoover, 1986).
Rapeseed meal.
Supplementation of diets with rapeseed meal decreased ruminal and total tract digestibility of NDF by 3 and 2 percentage units, respectively. This effect can be attributed to the higher concentration of indigestible NDF in rapeseed meal compared with other feed ingredients (data not presented). Low rumen ammonia concentrations are known to constrain microbial N synthesis (<3.3 mg/100 mL) and fiber digestion (<8.0 mg/100 mL; Hoover, 1986). In diets with no rapeseed meal supplementation, ruminal ammonia concentrations remained below 3.3 mg/100 mL for extended periods (6 and 3.5 h for diets S and SB, respectively; data not presented). Rapeseed meal supplementation increased rumen ammonia concentrations but had no beneficial effects on microbial protein synthesis or fiber digestion consistent with previous findings (Ahvenjärvi et al., 1999).
Ruminal N Metabolism
Microbial pools.
Variation in atom% excess of 15N between rumen microbial pools (Table 4) is consistent with previous studies. Martin et al. (1994) noted marked differences in 15N atom% between LAB, PAB, and protozoa (0.164, 0.112, and 0.094, respectively). Faichney et al. (1997) reported that the proportion of microbial N derived from rumen ammonia N was 65, 44 and 27% for LAB, PAB, and protozoa, respectively. These observations indicate that accurate measurements of microbial N flow leaving the rumen require establishing the contribution from each microbial fraction to total flow.
The use of particle and liquid phase markers allows digesta flow in particulate matter and liquid phases to be calculated. Thereby, microbial flow in each phase can be estimated from the respective chemical composition of microbial matter. Assuming fungi have a marginal contribution to microbial N flow (Faichney et al., 1997), representative samples of LAB, PAB, and protozoa are required. Representative sampling of PAB is probably the most questionable, because detachment of PAB is likely to be incomplete. Craig et al. (1987) reported that a combined procedure of chilling, use of surfactant, and saline extraction resulted in between 32 to 52% of PAB being recovered. Martín-Orúe et al. (1998) reported that various treatments caused detachment of between 59 and 68% of PAB, but total recovery was only between 19 and 22%.
In the present study, protozoal flow entering the omasal canal was determined by separating protozoa from a known volume of omasal canal digesta by centrifugation followed by rinsing with saline on a 17-µm polyester filter. The accuracy of this procedure is dependent on both recovery and the extent of contamination of the protozoal sample. After washing the ruminal particulate matter with saline, the vast majority of the protozoal mass can be expected to be transferred to the liquid phase (Coleman, 1985). Isolation of protozoa by filtration with 10- and 20-µm polyester bags has been shown to result in 83.1 and 68.1% recovery of protozoal numbers, respectively (Neill and Ivan, 1996). However, substantial bacterial contamination was observed when using 10-µm bags, whereas, use of 20-µm bags resulted in negligible contamination. In the present study, a 17-µm filter was chosen because it allowed proper filtration during the rinsing procedure. Because small protozoa are likely to escape through the filter more readily than large ones, recovery on the basis of protozoal mass should be greater than estimated by counting of protozoal numbers. Protozoal N flow determined in the present study is likely to be around or above 70% of that truly entering the omasal canal. Because NLAB flow was calculated using microbial N in the small particle and liquid phase that was not taken into account in protozoal N flow, the flow of NLAB is overestimated to the same extent as protozoal N flow is underestimated.
Previous studies have established that the majority of ruminal bacteria are associated with particulate matter (70 to 80%, Craig et al., 1987; 70%, Legay-Carmier and Bauchart, 1989; 74%, Hristov and Broderick, 1996). Despite greater proportions of PAB in ruminal microbial mass, due to higher passage rates of liquid than particulate matter, the proportion of LAB and PAB in microbial N leaving the rumen has been reported to be similar (Hristov and Broderick, 1996). In the present study, LAB, PAB, and protozoa contibuted to 67, 26, and 7%, respectively, of microbial N entering the omasal canal. Assuming that microbial mass is comprised of 25 and 75% of LAB and PAB, respectively, and fractional passage rates of 0.11 and 0.02 for the rumen liquid and particulate matter pools, respectively (Huhtanen and Jaakkola, 1992; Jaakkola and Huhtanen, 1992), LAB and PAB would have contributed to 65 and 35%, respectively, of bacterial N entering the omasal canal.
Mean microbial N and nonmicrobial N flows of 206 and 142 g/d, respectively, were observed in the current study (Table 5). Use of 15N enrichment determined in LAB alone would have resulted in flows of 190 and 157 g/d, respectively. Corresponding, estimates of 201 and 146 g/d would have been attained based on mean 15N enrichment of LAB and PAB. The latter indicate that ignoring the contribution of protozoa to total microbial flow was to a large extent compensated for by the overestimation of the proportion of PAB in microbial mass.
Supplementation of diets with barley increased NLAB flow suggesting that nonstructural carbohydrate-fermenting bacteria were primarily associated with small particulate matter and liquid. Michalet-Doreau et al. (2001) reported that a greater proportion of cellulolytic bacteria was associated with the solid compared with the liquid phase. However, the distinction between PAB and LAB is not necessarily equivalent to that between structural and nonstructural carbohydrate-fermenting bacteria. Currently, 9% of NDF entering the omasal canal was associated with small particles and liquid, indicating that a small proportion of particulate matter of cell wall origin was associated with these phases.
Barley.
Supplementation of diets with barley increased omasal canal NAN flow by 55 g/d (Table 5), attributed to increased NLAB (54 g/d), protozoal N flow (14 g/d), and decreased nonmicrobial N flow (-13 g/d). Because barley had no discernible effect on the efficiency of microbial protein synthesis, increased microbial N flow was entirely due to greater amounts of OM being truly digested in the rumen (12.5 vs 9.6 kg/d for barley and nonbarley diets, respectively).
Barley supplementation increased the proportion of LAB (0.65 vs 0.69) and protozoa (0.05 vs 0.10) in microbial N entering the omasal canal, but decreased that of PAB (0.30 vs 0.22). Supplementing grass hay with barley has been shown to increase the proportion of LAB (0.53 vs 0.68) and protozoa (0.05 vs 0.12), but reduce that of PAB (0.40 vs 0.18) in microbial N flow entering the duodenum of sheep (Faichney et al., 1997). The effect of barley supplementation on protozoal N flow is consistent with earlier studies indicating higher numbers of protozoa in rumen fluid associated with concentrate supplementation of grass silage diets (Chamberlain et al., 1985; Jaakkola and Huhtanen, 1992). Decreases in protozoal N content on barley diets can potentially be explained by dilution of protozoal cell contents by ingested starch particles (Hungate, 1966).
Barley supplementation increased the amount of N truly digested in the rumen to a greater extent than N intake (59 vs 46 g/d, respectively). This suggests that barley increased ruminal N degradability of the whole diet. The role of protozoa in ruminal N degradability was not assessed, but the increase in protozoal N flow suggests that barley stimulated increases in rumen protozoal mass. Previous studies have indicated more extensive degradation of N in the rumen of faunated than defaunated sheep (Jouany et al., 1988; Ivan et al., 2000).
Barley supplementation had little effect on the amount of N apparently digested in the rumen (67 vs 75 g/d for barley and nonbarley diets, respectively). Because apparent ruminal N degradability represents the net disappearance of dietary N from the rumen as ammonia lost through absorption and outflow, it appears that provision of readily digestible carbohydrates in barley did not substantially improve microbial capture of ruminal ammonia. Small differences between nonbarley and barley supplemented diets in the ratio of N to OM truly digested in the rumen (25.6 vs 24.5 g N/kg OM truly digested, respectively) are consistent with this conclusion. Furthermore, barley had no effect on urinary N excretion or rumen ammonia concentrations. However, the lower pH in the rumen of cows fed barley-supplemented diets may have decreased the rate of absorption, confounding the association between ammonia synthesis in and absorption from the rumen (Chamberlain et al., 1985; Siddons et al., 1985).
Supplementation of diets with barley increased omasal canal NAN flow by 54 g/d but had no effect on the amount of N excreted in urine. Therefore, amino acids absorbed from the intestine appeared to have been utilized at a higher efficiency for milk and body tissue protein synthesis. Assuming an intestinal amino acid digestibility of 85%, increases in the flow of amino acids entering the omasal canal (280 g/d; Korhonen et al., 2002) would be equivalent to 38 g/d (280 • 0.16 • 0.85) of absorbed amino acid N, that is consistent with the sum of milk protein N secretion and tissue N retention (37 g/d). A high apparent utilization of marginal increases in amino acid supply may be partly related to improvements in energy balance, that would reduce the dependence on energy derived from amino acid catabolism.
Rapeseed Meal.
Rapeseed supplementation increased N intake by 103 g/d (Table 5), the majority of which was degraded in the rumen (74 g/d). The major proportion of supplementary N degraded in the rumen was absorbed as ammonia (60 g/d) and subsequently excreted in urine (48 g/d). Substantial losses of additional N in the rumen are consistent with increases in the ratio of N to OM truly degraded in the rumen (27.7 vs 22.3 g N/kg OM truly digested for rapeseed and nonrapeseed diets, respectively). This suggests that the supply of fermentable energy available to rumen microbes constrained the assimilation of supplementary N for microbial protein synthesis. On a theoretical basis, efficient capture of dietary N is dependent on the amount of N relative to OM degraded in the rumen being equivalent to or less than the efficiency of microbial N synthesis (18.5 g N/kg OM truly degraded).
Supplementation of diets with rapeseed meal increased omasal canal total NAN flow by 43 g/d, that can be primarily attributed to higher nonmicrobial N (29 g/d) and NLAB flow (18 g/d). The amount of amino acids entering the omasal canal increased by 277 g/d (Korhonen et al., 2002) suggesting that amino acid N absorbed from the small intestine increased by 38 g/d (277 • 0.16 • 0.85 = 38). Because the sum of milk protein N yield and N retention (47 g/d) increased to a greater extent than calculated amino acid N absorption, it appears that either utilization was overestimated or rapeseed meal improved the utilization of absorbed amino acids overall.
Growth of nonstructural carbohydrate-fermenting bacteria has been suggested to be stimulated in the presence of peptides and amino acids in the rumen (Russell et al., 1992). However, despite considerable increases in the amount of protein degraded in the rumen, rapeseed meal had no effect on the efficiency of microbial N synthesis, either alone or in combination with barley. This finding is consistent with previous findings (Ahvenjärvi et al., 1999), suggesting that amino acid supply from silage satisfied rumen microbe amino acid and peptide requirements. This suggestion is supported by the observation that 76.7 percentage units of total silage N were in the form of amino acids (Korhonen et al., 2002).
Milk Yield
Consistent with previous studies (Huhtanen, 1998), supplementation of silage diets with barley and rapeseed meal markedly improved milk yield (2.3 and 3.0 kg/d, respectively). The effect of barley could be explained by an increase in digestible OM intake (2.5 kg/d), that supplied more fermentable energy for ruminal microbes, and thereby, increased the supply of energy and protein to the host animal. In contrast, rapeseed meal had much smaller effects on digestible OM intake (1.0 kg/d), such that benefits in milk production can be attributed to increases in the supply of rumen undegraded protein.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication October 22, 2001. Accepted for publication April 19, 2002.
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I. R. Ipharraguerre, S. M. Reynal, M. Lineiro, G. A. Broderick, and J. H. Clark A Comparison of Sampling Sites, Digesta and Microbial Markers, and Microbial References for Assessing the Postruminal Supply of Nutrients in Dairy Cows J Dairy Sci, April 1, 2007; 90(4): 1904 - 1919. [Abstract] [Full Text] [PDF]
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A. F. Brito, G. A. Broderick, J. J. O. Colmenero, and S. M. Reynal Effects of Feeding Formate-Treated Alfalfa Silage or Red Clover Silage on Omasal Nutrient Flow and Microbial Protein Synthesis in Lactating Dairy Cows J Dairy Sci, March 1, 2007; 90(3): 1392 - 1404. [Abstract] [Full Text] [PDF]
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A. F. Brito, G. A. Broderick, and S. M. Reynal Effect of varying dietary ratios of alfalfa silage to corn silage on omasal flow and microbial protein synthesis in dairy cows. J Dairy Sci, October 1, 2006; 89(10): 3939 - 3953. [Abstract] [Full Text] [PDF]
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S. M. Reynal, G. A. Broderick, and B. Larget Short communication: effectiveness of sample duplication to control error in ruminant digestion studies. J Dairy Sci, September 1, 2006; 89(9): 3501 - 3504. [Abstract] [Full Text] [PDF]
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J. L. Firkins, A. N. Hristov, M. B. Hall, G. A. Varga, and N. R. St-Pierre Integration of Ruminal Metabolism in Dairy Cattle J Dairy Sci, March 1, 2006; 89(e_suppl_1): E31 - E51. [Abstract] [Full Text] [PDF]
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S. M. Reynal and G. A. Broderick Effect of Dietary Level of Rumen-Degraded Protein on Production and Nitrogen Metabolism in Lactating Dairy Cows J Dairy Sci, November 1, 2005; 88(11): 4045 - 4064. [Abstract] [Full Text] [PDF]
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S. M. Reynal, G. A. Broderick, and C. Bearzi Comparison of Four Markers for Quantifying Microbial Protein Flow from the Rumen of Lactating Dairy Cows J Dairy Sci, November 1, 2005; 88(11): 4065 - 4082. [Abstract] [Full Text] [PDF]
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J. T. Sylvester, S. K. R. Karnati, Z. Yu, C. J. Newbold, and J. L. Firkins Evaluation of a Real-Time PCR Assay Quantifying the Ruminal Pool Size and Duodenal Flow of Protozoal Nitrogen J Dairy Sci, June 1, 2005; 88(6): 2083 - 2095. [Abstract] [Full Text] [PDF]
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I. R. Ipharraguerre and J. H. Clark Impacts of the Source and Amount of Crude Protein on the Intestinal Supply of Nitrogen Fractions and Performance of Dairy Cows J Dairy Sci, May 1, 2005; 88(e_suppl_1): E22 - E37. [Abstract] [Full Text] [PDF]
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A. Sairanen, H. Khalili, J. I. Nousiainen, S. Ahvenjarvi, and P. Huhtanen The Effect of Concentrate Supplementation on Nutrient Flow to the Omasum in Dairy Cows Receiving Freshly Cut Grass J Dairy Sci, April 1, 2005; 88(4): 1443 - 1453. [Abstract] [Full Text] [PDF]
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H. G. Bateman II, J. H. Clark, and M. R. Murphy Development of a System to Predict Feed Protein Flow to the Small Intestine of Cattle J Dairy Sci, January 1, 2005; 88(1): 282 - 295. [Abstract] [Full Text] [PDF]
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