J. Anim. Sci. 2002. 80:2704-2716
© 2002 American Society of Animal Science
Apparent ruminal degradation and rumen escape of soluble nitrogen fractions in grass and grass silage administered intraruminally to lactating dairy cows1
H. Volden*,
L. T. Mydland* and
V. Olaisen
* Department of Animal Science, Agricultural University of Norway, P.O. Box 5025, NO-1432 Ås, Norway and
and
Department of Chemical Engineering, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway
Correspondence:
phone: +47 64 94 80 00; fax: +47 64 94 79 60; E-mail:
harald.volden{at}ihf.nlh.no.
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Abstract
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The main objective of this study was to investigate in vivo ruminal degradation and rumen escape of soluble N fractions in grass and grass silage. Soluble protein and long-chain peptides (PLP), small peptides (SP) and free AA (FAA) were obtained from fresh grass and grass silages fertilized with different levels of N. Soluble extracts from the forages were pulse dosed into the rumen of three cannulated lactating dairy cows, and a simple or complex model was used to examine the kinetics of the soluble N fractions in the rumen. When soluble extracts from silage were investigated, pulse dosages of total nonammonia N (NAN) were 21, 27, and 32 g, while for fresh grass only dosages of 20 g were ruminally administered. In the silage extracts, mean proportions of PLP-N, SP-N, and FAA-N in the NAN were 30, 52, and 18%, respectively, whereas in the fresh grass the corresponding values were 67, 20, and 13%. From silage extracts, all three soluble N fractions showed a linear decrease (P < 0.05) in degradation rate and an increase (P < 0.05) in ruminal escape with increasing dosage. In silage, mean degradation rates, parameterized from the complex model, were 230, 214, and 334%/h for PLP-N, SP-N, and FAA-N, respectively, and the ruminal escape was highest (P < 0.05) for SP-N (11.2% of dose) and lowest (P < 0.05) for FAA-N (5.0% of dose). No differences in degradation rate and ruminal escape between fresh grass and silage were observed. However, the proportion of N dose converted to ammonia was only 24% in the fresh grass, whereas for the silages a mean value of 76% was found. From this study, it is concluded that a significant amount of dietary soluble N escapes ruminal degradation, and thus contributes to the intestinal AA supply. Moreover, if the main aim is to study degradation kinetics of individual N fractions, a complex model should be used in the evaluation. This model can also be used to study ruminal synchronization of N and energy for microbial growth.
Key Words: Dairy Cows • Grass Silage • Nitrogen • Protein Degradation • Rumen Digestion
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Introduction
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Proteins consumed by ruminants are partly broken down to peptides, AA, and finally to ammonia by the bacteria and protozoa in the rumen. When protein degradation is rapid, the rumen microbes are unable to utilize all the peptides, AA, and ammonia; thus, more dietary N will be degraded than that which is used for microbial protein synthesis. However, Chen et al. (1987a) showed that small peptides accumulated in the rumen after feeding and thereby escaped ruminal degradation, and Volden et al. (1998) observed that on average 20% of intraruminally administered free AA (FAA) escaped the rumen. These results indicate that parts of the soluble dietary N are not degraded in the rumen and can therefore supply the host animal with AA. Proteins in grass and grass silage are very susceptible to microbial breakdown and are often poorly utilized by the rumen microbes (Beever, 1993). During ensiling, the protein has been impaired by extensive proteolysis, and a high proportion of the N in grass silage is in the form of soluble nonprotein N (NPN)—mainly small peptides and FAA (McDonald, 1981). Therefore, to improve the accuracy of determination of the protein value of grass and grass silages, it is important to know how much of the dietary soluble N escapes the rumen. Moreover, lower efficiency in microbial protein synthesis on grass silage than in grass diets may be due to the asynchronous release of N and carbohydrates in grass silage (Siddons et al., 1985), and, therefore, to identify the potential benefits from improved synchronization, it is vital to know the rate of degradation of soluble protein, peptides, and FAA in grass and grass silages. The first objective of this study was to estimate in vivo ruminal degradation rate and rumen escape of soluble N fractions in grass and grass silage. The second objective was to develop a mechanistic model of soluble N degradation in the rumen and compare this with a simple model.
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Materials and Methods
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Animals, Feeding, and Management
Three Norwegian Cattle multiparous dairy cows, weighing 642 ± 15 kg and yielding 34.1 ± 1.4 kg/d of milk at the start of the experiment were used. They were fitted with a ruminal cannula of 10 cm i.d. (Bar-Diamond, Parma, ID). Cows were housed individually in metabolism stalls, had free access to water, and were fed a total mixed ration (TMR) consisting of 58% concentrate mixture and 42% grass silage on a DM basis (Table 1
). Daily DMI was fixed to 20 kg/d to avoid feed refusals. The grass silage originated from a mixture of timothy, meadow fescue, and red clover, and was first cut, prewilted, and ensiled with a formic acid based additive. The TMR was given in four equal meals at 0600, 1500, 1800, and 2200. Feed not eaten at 1000 was removed and given with the meal at 1500. The cows were milked at 0630 and 1530.
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Table 1. Ingredients and chemical composition of the concentrate mixture and chemical composition of the grass silage used in the total mixed ration
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All cows were cared for according to laws and regulations controlling experiments on live animals in Norway (i.e., the Animal Protection Act of December 20, 1974, and the Animal Protection Ordinance concerning Experiments on Animals of January 15, 1996).
Soluble Nitrogen Preparation, Treatments, Experimental Design, and Ruminal Samples
The soluble N fractions were obtained from four forages: (1) Fresh grass from first cut consisting of a mixture of timothy and meadow fescue fertilized with 60 kg N/ha (GN1); (2) grass silage (SN1) ensiled from the same sward as GN1; (3) grass silage from first cut consisting of a mixture of timothy and meadow fescue fertilized with 180 kg N/ha (SN3); and (4) grass silage from second cut consisting of a mixture of timothy, meadow fescue and red clover with no N fertilizer (SN0). The proportion of red clover was 63% on fresh weight basis. The first cut was harvested at beginning of heading of the timothy. The grass was chopped to a 2.0-cm theoretical length of cut, and the fresh grass was stored at -18°C until extraction of soluble N. The silages were ensiled in 100-L laboratory silos with a formic acid-based additive using 0.7 kg/100 kg fresh material. For each material, 300 kg were stored prior to extraction of soluble N. Chemical composition of the grass and the grass silages are presented in Table 2. To obtain samples of soluble nonammonia N (NAN) for ruminal administration, the material was first crushed in a meat blender for 15 min and then stored at 39°C for 1 h. Thereafter, the material was placed in a steel cylinder (inner diameter of 35 cm) with a perforated bottom stopper, and a liquid phase was pressed out using a hydraulic press with a pressure of 255 kPa. The extracts were evaporated at 45°C for 20 h to reduce the liquid volume and thus to increase the N concentration. The concentration of NAN in the liquid extracts before and after evaporation is presented in Table 3. The NAN in the extracts was separated into protein and long-chain peptides (PLP), short-chain peptides (SP), and FAA on the basis of analysis. The extracts were administered intraruminally as pulse doses to study rumen kinetics, and for the silages dosages of 21, 27, and 32 g of NAN were given. For GN1 only dosages of 20 g of NAN were managed due to shortage of material. Before being administered to the rumen, each dosage was diluted with tap water to a final volume of 3.25 L. To determine rumen liquid pool size and liquid passage rate, 12 g of Co-EDTA (Udén et al., 1980) was mixed together with the extract and administered simultaneously to the rumen. A preliminary test showed that postfeeding values of ruminal soluble N-fractions had decreased to prefeeding values at 1000. The pulse dosages were therefore given at that time to reduce interference from the basal diet. When ruminal N kinetics in silage extracts were studied, treatments were arranged as a Latin Square split plot design. For each silage type the cows received each dosage in random order according to a 3 x 3 Latin Square design. A total of nine dosages were given for each silage type (3 cows x 3 dose sizes), and each cow received one dosage every day. One experimental period lasted 24 h. When extract from GN1 was examined, all cows received only one dosage on the same day.
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Table 2. Chemical composition and in situ CP degradation characteristics of the experimental forage used for extraction of soluble N
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Table 3. Concentrations of protein and long-chain peptide N, short-chain peptide N, free AA N, and total nonammonia N in the liquid extracts before and after evaporation
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Ruminal samples (250 mL) were taken just before administration of soluble N and thereafter 15, 30, 45, 60, 75, 90, 120, 150, 180, 240, and 300 min after dosage. Additional samples to determine ruminal Co concentrations were taken 7, 9, 11, 13, and 24 h after dosage. The samples were immediately placed on ice to reduce microbial and enzymatic activity, then filtered through an 80-µm nylon filter to remove large digesta particles. Thereafter, the samples were centrifuged at 27,000 x g for 15 min at 4°C to remove small digesta particles and microbes. After centrifugation, subsamples were taken from the supernate to measure the concentration of total N, FAA-nitrogen (FAA-N), ammonia nitrogen (NH3-N) and Co, and to precipitate with trichloroacetic acid (TCA). To precipitate protein and long chain peptides (>10 units; Greenberg and Shipe, 1979) 10 mL of 50% TCA was added to 50 mL of the supernate. This solution was incubated at room temperature for 1 h, and then centrifuged at 27,000 x g for 15 min. To determine small peptides, 2.5 mL of the supernate was hydrolyzed with 6M HCl at 110°C for 24 h. The increase in FAA-N was used as a measure of peptide amino N and the concentration of SP-nitrogen (SP-N) was estimated from the difference between FAA-N concentration in the hydrolysate and the FAA-N concentration in the sample prior to hydrolysis and TCA precipitation. The concentration of PLP-nitrogen (PLP-N) was estimated as the difference between total NAN prior to precipitation with TCA and the amount of FAA-N in the hydrolysate. Two days prior to soluble N dosage, ruminal samples were obtained to study the effect of feeding on ruminal N concentrations. Samples were taken just before feeding at 0600 and thereafter 15, 30, 45, 60, 75, 90, 120, 150, 180, and 240 min postfeeding. Control samples used in the calculation of ruminal net N concentrations were obtained 1 d prior to dosage and followed the same regime of sampling as described for the samples taken at dosing.
Ruminal degradation characteristics of CP in lyophilized forage samples were measured using the in situ method. Nylon bags were incubated in the rumen for 2, 4, 8, 16, 24, 48, and 72 h. In situ water soluble CP (0-hour disappearance) was obtained by washing the nylon bags in a domestic washing machine. Feed preparation, nylon bags, rumen incubation program, and washing and drying procedures were completed as described by Volden and Harstad (1995).
Model Description and Calculations
Two models were developed to estimate the ruminal degradation rate and the rumen escape of soluble N fractions. In both models, extracellular soluble N is divided into three pools: PLP-N, SP-N, and FAA-N. In the complex model, NH3-N is additionally included. In the first model, the simple one, each pool of soluble NAN is treated individually (Figure 1) and ruminal degradation rates of PLP, SP, and FAA are estimated assuming first order degradation kinetics. The second model is more complex and takes into account that ruminal degradation of soluble N fractions follows interacted sets of reactions. A schematic description of the model is shown in Figure 2, and all processes are assumed to be of first order. The first step is an extracellular degradation of PLP into SP:
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Figure 1. Flow diagram describing the simple model for prediction of ruminal degradation of dietary soluble N fractions. For explanation of the model, see text.
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Figure 2. Flow diagram describing the complex model for prediction of ruminal degradation of dietary soluble N fractions. The intracellular (IC) pools are inside the dashed rectangle. For explanation of the model, see text.
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 | [1] |
where CPLP is the N concentration (mg/L) of PLP, k1 (h-1) is the degradation rate and kp (h-1) is the liquid passage rate. The next step is an active transport of SP and FAA into the microbes:
 | [2] |
 | [3] |
where CSP and CFAA is the N concentration (mg/L) of the extracellular SP and FAA, k2 is the transport rate of SP and k3 of FAA into the microbes. A transport of FAA out of the microbes has also been reported (Udén, 2000), and k3 therefore represents the net transport of FAA into the microbes. The peptides and AA are hydrolyzed intracellularly, and microbial protein is synthesized from AA, NH3, and possibly peptides. Excess NH3 not used for microbial protein synthesis is transferred out of the microbes, where it is either absorbed through the rumen wall or follows the liquid fraction out of the rumen. Ammonia flows are described by the following equation:
 | [4] |
where CAm is the extracellular NH3-N concentration, rate variable k4 represent the absorption rate of ammonia from the rumen, and f is the fraction of dietary N degraded into NH3. An f value close to zero indicates high efficiency in converting ammonia into microbial protein synthesis, whereas an f value close to one indicates low efficiency.
The set of linear differential equations (1 to 4) were solved analytically, and the concentration profiles are given by
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 | [6] |
 | [7] |
 | [8] |
where
, 
, 
are the dosage concentrations (mg N/L) of PLP, SP, and FAA in rumen, and 
is the concentration of ammonia at the time of dosage administration. The background concentrations, 
, 
, 
, and 
, were assumed to be constant throughout the sampling period.
The ruminal escape of PLP-N, SP-N, and FAA-N was estimated by integration of the liquid residence time distribution (RTD) equation and the equations describing the profiles of soluble N concentrations (Eqs. [5]
, [6], and [7]). In the equations describing the concentration profiles, the passage rates were set to zero and the background concentration terms were removed. Using a mixing compartment RTD model, the ruminal escape values (%) were calculated by:
 | [9] |
 | [10] |
 | [11] |
The liquid passage rate was estimated from the ruminal Co data using a mixing compartment model: 
, where
is the Co concentration (mg/L) at t = 0 and kpis the liquid passage rate (h-1). The ruminal pool size was estimated from the mass of Co in the dosage (mCo, mg) and the estimated Co concentration at t = 0: 
.
The parameters in Eqs. [5] through [8] were estimated simultaneously by a constrained nonlinear optimization algorithm implemented in Matlab (MathWorks, Inc., NA).
The kinetics of in situ CP degradation were calculated according to the following exponential model: D(t) = a + b x (1 - e-ct), where D(t) is the percentage of CP degraded from the bag at time t of the soluble (a) and insoluble (b) CP fractions and c is the fractional degradation rate of b. The nonlinear parameters were obtained by using the NLIN procedure of SAS (SAS Inst. Inc., Cary, NC).
Chemical Analysis
Feeds were analyzed for DM, ash, crude fat, and Kjeldahl N according to the procedures described by AOAC (1980). The contents of NDF and ADF in feeds were measured using the method described by Van Soest et al. (1991). Starch in the concentrate mixture was analyzed according to the method of McCleary et al. (1994). The Co concentrations in ruminal liquid samples were estimated by atomic absorption spectrometry (GBC 906AA; Scientific Equipment PTY Ltd., Dandenong, Victoria, Australia), and an acetylene flame was used in the determination. Ruminal liquid samples and soluble forage extracts were analyzed for NH3-N according to the procedure of Tingwall (1978). Total N was determined by the semi-micro-Kjeldahl procedure (75-mL digestion flasks) using copper as a digestion catalyst (AOAC, 1980), and total N was analyzed by the Tecator Flow Injection Analysis (Tecator, 1989). Buffer soluble N in lyophilized forage was measured by extraction in a borate-phosphate buffer according to the method of Licitra et al. (1996). Concentration of FAA-N in ruminal liquid samples before precipitation with TCA and before HCl hydrolysis was determined by the cadmium-ninhydrin method (Baer et al., 1996). This method was chosen because preliminary tests showed that compared to ninhydrin this method gave a low response to di and tri peptides. Cadmium-ninhydrin reagent: 0.8 g ninhydrin (Sigma, N 4876) was dissolved in 80 mL 96% ethanol, 10 mL of concentrated acetic acid, and 1 mL CdCl2. The reagent was stored in a brown glass bottle at room temperature. Analytical procedure: 60 µL of sample was dissolved in 240 µL of deionizied water. Thereafter, 600 µL of the cadmium-ninhydrin reagent was added, thoroughly mixed, and incubated in a water bath at 85°C for 10 min. After incubation, the solution was cooled on ice for 5 min and the content of FAA was subsequently analyzed on a spectrophotometer at 492 nm. The concentration of FAA after HCl hydrolysis was assayed by a ninhydrin method (OJ, 1998) using a ninhydrin reagent kit (Amersham Pharmacia Biotech, 80-2038-07). First 4.45 mL of the ninhydrin reagent was added to 50 µL of sample and thoroughly mixed. After incubation in a water bath at 90°C for 5 min, the samples were cooled and the content of FAA after hydrolysis was subsequently analyzed on a spectrophotometer at 570 nm. In the analysis of FAA a 50:50 mixture of alanine and leucine was used as stock standard.
Statistical Analyses
Statistical analysis was conducted with the general linear models procedure of SAS. When data from silage extracts were analyzed, the model sum of squares were divided into silage type (n = 3), soluble N fraction (n = 3), animal (n = 3), period (n = 3), and dosage (n = 3) plus interactions of silage type x dosage, silage type x soluble N-fraction, silage type x dosage x soluble N fraction, and animal x period x dosage x silage type. When to detect differences between silage types and dosages, the animal x period x dosage x silage type was used as the error term. Orthogonal contrasts were used to separate mean differences of silage type and soluble N fractions. Orthogonal polynomials were used to test linear and quadratic responses to increased soluble N dosages. When studying the effects between fresh grass and grass silage from the same sward the statistical model included animal, soluble N fraction and forage type plus interactions of soluble N-fraction and forage type. Orthogonal contrasts were used to separate mean differences of forage type. Differences were considered statistically significant when P < 0.05, and trends were considered to exist when 0.05 < P < 0.10.
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Results and Discussion
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Chemical Composition of the Forage and the Extracts
A high proportion of red clover in SN0 gave a lower NDF content than in SN1 and SN3, whereas the CP content in SN0 was at the same level as in the forage (SN3) fertilized with 180 kg N/ha (Table 2). A higher N fertilization resulted in a higher CP content in SN3 than in GN1 and SN1. No difference in NDF content between SN1 and SN3 was observed. However, the content of NDF in GN1 was 10 percentage units higher than in SN1, whereas small differences were observed in ADF content. These results demonstrate that parts of the hemicellulose fraction in grass are used as a substrate in the ensiling process. The proportion of soluble N in the fresh forage was lower than in the silages; this is in agreement with previous reports (Albrecht and Muck, 1991; Petit and Tremblay, 1992). The silage containing red clover (SN0) had 58% lower buffer soluble N than the silage containing only grass, which are similar to what was found by Vik-Mo (1989). Ruminal degradation characteristics of CP measured in situ are presented in Table 2. The values of in situ water soluble CP showed, in general, good agreement with values found for buffer soluble N. The degradation rate of the slowly degradable fraction was lower in the fresh grass than in the silage (GN1 vs SN1).
The NAN characteristics of the forage extracts are presented in Table 3. The higher proportions of SP-N and FAA-N in the silages than in the grass can be explained by the conservation process. Plant enzymes and microbial proteolysis during ensiling will result in an extensive conversion of soluble proteins to NPN fractions (McDonald, 1981). In the non-evaporated silage extracts, the average proportions of SP-N and FAA-N in the total NAN were 52 and 18%, respectively, while in the fresh grass the corresponding values were 20 and 13%. The proportion of PLP observed for the silages (30%) is in agreement with Carpintero et al. (1979) who found a proportion of 36% in grass silage ensiled with formic acid as an additive. Moreover, using tungstic acid as a precipitant, Messman et al. (1994) found that the proportion of NPN in the soluble N of grass silage on average was 63%, which is in good agreement with the mean value observed in the present study (69%). Fresh grass has a higher content of water-soluble carbohydrates than grass silage (Cushnahan et al., 1995) and this may explain the lower NAN concentration in the extract from GN1 than from SN1. Preliminary tests showed that to obtain a sufficient increase in ruminal NAN concentrations after dosage it was necessary to increase the NAN concentration in the extracts by evaporation of water. For silage extracts, the evaporation process resulted in small effects on the ratios between the soluble N fractions (Table 3). However, for the GN1, evaporation increased the ratio of especially small peptides but also for FAA, indicating protease and peptidase activity from plant enzymes.
Ruminal Concentrations of Soluble N fractions after Feeding the Basal Diet
Concentrations in the rumen of NH3-N, PLP-N, SP-N, and FAA-N when cows were fed the basal diet are presented in Figure 3. Concentrations of SP-N, FAA-N, and NH3-N increased rapidly after feeding and for FAA-N it reached maximum after 45 min while for NH3-N and SP-N maximum levels were achieved 75 min postfeeding. After reaching their maximum levels, the concentrations declined rapidly and within a 3-h period after feeding the values had almost returned to prefeeding levels. The concentrations of FAA-N observed both before and after feeding are at the same levels as found by Volden et al. (2001) when FAA analysis was based on individual amino acid measurements. The three-fold increase in peptide concentration observed 75 min after feeding is lower than values observed by Williams and Cockburn (1991). In an experiment with steers fed different protein sources, Williams and Cockburn (1991) observed an increase in peptide-N concentrations from 4 mg/L before feeding to 88 mg/L 1 h after feeding. The average ruminal peptide-N concentration in their study (96 mg N/L) was lower than 170 mg/L, which was observed in the present experiment. Moreover, in a study with lactating dairy cows fed 21 kg DM/day, Robinson et al. (1999) observed a mean ruminal peptide-N concentration of 124 mg/L. However, their ruminal sampling regime was much wider in time than what was used in the present study and may explain the lower values. Although the different levels of ruminal peptide concentrations observed between different studies are an effect of different diets, they are probably also a result of different analytical methods to determine peptides (Wallace, 1996; Wallace et al., 1997). In a study with lactating dairy cows fed alfalfa silage at a feeding level of 17.1 kg DM/day, Hristov and Broderick (1996) measured a dietary NAN concentration in ruminal liquid of 409 mg/L, which is about 30% higher than that observed in the present study.
The major peptidase activity in the rumen is aminopeptidase, which is characterized by dipeptides rather than single FAA being cleaved from the peptide chain (Wallace et al., 1997). This provides evidence that SP rather than FAA are the main end product of proteolysis and which may explain the low concentrations of FAA in ruminal liquid as observed in the present study and by Broderick et al. (1981) and Volden et al. (2001).
Ruminal Degradation Rate and Rumen Escape of Dietary Soluble N-fractions
An example of soluble N profiles in the rumen after administration of a pulse dose of dietary soluble N is shown in Figure 4. The figure represents mean values from three cows and the three dosages of treatment SN1. All three NAN-fractions decreased rapidly and FAA-N and PLP-N reached equilibrium after 2 h, whereas for SP-N equilibrium was reached approximately 3 h post-dosing. The profiles presented in the figure were very typical for all treatments, indicating that all three dietary soluble N fractions were very rapidly degraded. Similar profiles have also been observed in experiments with steers or sheep given casein (Mangan, 1972; Broderick and Wallace, 1988) or fraction I leaf protein (Nugent and Mangan, 1981). However, in the present study we expected another profile for NH3-N, with a slower increase and thus a maximum concentration at a later stage after pulse dosing. In most of the cases, highest NH3-N concentration was achieved 15 min after dosing, and thereafter declining in a form similar to what was observed for SP-N.
For 6 out of 30 pulse doses, Co concentration measured 15 min postdosing showed higher concentrations than expected. This implies an incomplete ruminal mixing, and, thus, a risk of overestimating the concentration of soluble N-fractions at the first sampling time. These high values were not removed from the parameterization of the degradation models, although they may give an overestimation of the degradation rate.
Mean fractional liquid passage rate was 16.6 (± 0.31) %/h and not affected by any treatments (results not shown). Ruminal degradation rate and rumen escape of dietary soluble N fractions in silage administered to the rumen at three dosages is presented in Table 4. No interaction was observed between soluble N fraction and dose size, and the degradation rate decreased linearly (P < 0.05) with increased dosage. When degradation rate was calculated from the simple model, ruminal NAN concentrations were corrected for ruminal outflow only. The degradation rate was highest (P < 0.05) for FAA-N and lowest (P < 0.05) for SP-N, and across grass silage type and dosages degradation rate of FAA-N was more than twofold higher than for SP-N. When the complex model is used in the calculations, the soluble N fractions will interact with each other, and this will affect the predicted results. In this model, PLP is first degraded to SP, and then both intracellular SP and FAA are partly degraded to NH3. Therefore, use of the complex model to calculate degradation rate will have greatest impact on the predicted values of SP because there is a substantial inflow of SP from the degradation of PLP. When calculated according to the complex model, the degradation rate of FAA-N was still higher (P < 0.05) than for PLP-N and SP-N. However, the average SP-N degradation rate was 69% higher when estimated from the complex model than from the simple model, which resulted in no difference in degradation rate between PLP-N and SP-N. The degradation rates of the different grass silages were generally higher for the complex model than for the simple model (Table 4 and 5). In these feeds, there was a large increase in NH3 concentration the first 15 min after dosing. Therefore, the degradation rates were higher when NH3 was incorporated in the models. The difference in degradation rate between the simple and complex model was much smaller for the fresh grass where the increase in NH3 concentration was smaller (Table 6).
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Table 4. Rumen fractional degradation rate and ruminal escape of intraruminally administered soluble N fractions from grass silage in dairy cows at three levels of dosage.a Parameterized according to a simple or complex model for describing ruminal degradation of soluble dietary N fractions
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Table 5. Rumen fractional degradation rate and ruminal escape of intraruminally administered dietary soluble N extracted from different grass silage typesa. Parameterized according to a simple or complex model for describing ruminal degradation of soluble dietary N fractions
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Table 6. Rumen fractional degradation rate and ruminal escape of intraruminally administered soluble N fractions from grass and grass silage harvested from the same sward.a Parameterized according to a simple or complex model for describing ruminal degradation of soluble dietary N fractions
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The observed degradation rate of PLP-N was in agreement with values found for fraction I leaf protein (Nugent and Mangan, 1981; Nugent et al., 1983). Fraction I leaf protein is the main soluble protein of plant leaves, and it is a linear protein where the molecule is held together with hydrogen bounds (Nugent et al., 1983). Nugent and Mangan (1981) observed an average initial degradation rate of 3.3 mg protein-N/L per min when sheep were fed a diet of hay and concentrate, whereas a value of 30 mg protein-N/L per min was observed when a fresh alfalfa diet was fed. In the present experiment, an average value of 8.5 mg PLP-N/L per min was found when calculated from the complex model. Moreover, the values observed in our study are also similar to values observed in studies where casein was administered intraruminally (Mangan, 1972; Nugent et al., 1983).
The degradation rate of peptides is dependent on their AA composition, AA sequence, and the nature of the protein in the diet (Wallace et al., 1990; Wallace et al., 1997). Moreover, which form, SP or FAA, that is most preferentially absorbed through the microbial cell membrane is rather obscure. Experiments have indicated that it is dependent on the diet composition, and thus on the microbes presented in the rumen (Wright, 1967; Armstead and Ling, 1993; Ling and Armstead, 1995). Consequently, dietary conditions may affect the degradation rate of SP. Chen et al. (1987a) found that peptides accumulated in the rumen when soybean meal was fed to dairy cows, and Broderick and Wallace (1988) observed a ruminal accumulation of peptides when casein was fed to sheep. However, Nugent and Mangan (1981) found no accumulation of peptides when they studied rumen proteolysis of fraction I leaf protein from alfalfa. In the present study, degradation rate of SP-N was slower than for PLP-N when calculated from the simple model. In addition, independent of model used, degradation rate of SP-N decreased with increased dosage. This indicates a transient accumulation of small peptides during proteolysis of PLP. Chen et al. (1987b) suggested that microbial uptake of peptides, rather than the rate of cleavage of the polypeptide chain, is the rate-limiting step in protein degradation. Consequently, a transient accumulation of soluble N-fractions may indicate that parts of these fractions have an opportunity to escape the rumen. Decreased degradation rate with increased dosage was also observed for PLP-N and FAA-N, and this explains why ruminal escape increased (P < 0.05) with increased dosage (Table 4). Across grass silage type and dosages, the mean values of rumen escape of dietary soluble N-fractions were highest for SP-N and lowest for FAA-N. When calculated from the simple model a mean value of 9.2% of the ruminally administered NAN escaped the rumen, whereas for the complex model the corresponding value was 7.8%. More than 95% of the administered FAA had disappeared from the rumen within the first hour after dosing, which resulted in a very high estimated degradation rate. Volden et al. (1998) studied degradation kinetics of intraruminally administered lysine, methionine, and threonine in lactating dairy cows, and found an average degradation rate of 101%/h when cows were fed at high feeding level, which is considerably lower than the values observed in the present study. However, they demonstrated that the degradation rate was different between individual AA and affected by level of AA dosage. In a study with dry cows, Velle et al. (1998) observed large variation among AA in degradation rate and that the composition of the ruminally administered FAA mixtures affected the amount of AA flowing out of the rumen. Moreover, experiments have shown that diet composition (Sulu et al., 1989) and feeding level (Volden et al., 1998) may affect FAA utilization, and this may in part explain the differences in degradation rate and ruminal escape observed between studies.
Choi et al. (1999) studied ruminal escape of soluble N fractions in dairy cows fed diets of grass silage and barley supplemented with rapeseed meal. They observed that of the total soluble N-fraction escaping the rumen, the relative contributions from FAA, SP, and PLP were 13, 63, and 24% respectively. Using a combination of average dose size, ratio of soluble N-fractions in the extracts, and ruminal escape calculated from the complex model, corresponding values observed in the present study were 10, 65, and 25%, respectively. These two studies show that small peptides are the most dominating soluble N-fraction leaving the rumen in grass silage based diets.
Independent of model used for parameterization, the degradation rate was lowest (P < 0.01) and ruminal escape highest (P < 0.05) for SN1 (Table 5). Predicted from the simple model, SN0 showed the highest (P < 0.05) degradation rate, and the same numerical differences were observed with the complex model, although the values were not significantly different. The lower degradation rate found for SN1 could mostly be explained by the differences in PLP-N and SP-N degradation rates, which on average were 91 and 57% lower than for SN0 and SN3, respectively, when measured from the complex model. It is difficult to explain the slower degradation rate, but at harvest the stem:leaves ratio was higher for SN1 than for SN3, and this may have affected protein structure, peptide length and the amino acid composition of the PLP and SP fraction. Nugent et al. (1983) studied degradation of different soluble proteins and suggested that structural folding of the protein molecule and the number of disulphide bridges and cross-links explained the differences in protein degradation. Moreover, Cooper and Ling (1985) showed that peptide uptake in bacteria was affected by the peptide size, and Wallace et al. (1990) showed that composition of the small peptides affected the degradation rate. Chen et al. (1987b) observed that hydrophobicity of the peptides determined their rate of degradation and that hydrophilic peptides were more rapidly taken up by the bacteria than hydrophobic peptides. In the present study, peptide-N was estimated by the reaction with ninhydrin before and after acid hydrolysis, and this method gives no indication of the type of peptide present. It is therefore difficult to explain the differences in peptide degradation rate observed between the silage extracts in this study.
Across soluble N fractions, dosage, and model mean escape (% of dose) of soluble dietary NAN in the silages was 8.5% and because of the slower degradation rate, the ruminal escape was higher for SN1 than for SN0 and SN3. Studying dietary soluble NAN flow into the omasum of dairy cows fed grass silage-based diets, Choi et al. (1999) found that on average 8.8% escaped the rumen. Recalculations of the data of Hristov and Broderick (1996) showed that when feeding alfalfa silage to lactating cows, 5.5% of the dietary NPN (corrected for NH3) flowed out of the rumen. Moreover, Chen et al. (1987a) found that a significant amount of peptides escaped the rumen of dairy cows, and according to Broderick and Wallace (1988) this contributed 4.7% of the total ruminal NAN outflow. Moreover, when measuring FAA outflow, Campbell et al. (1997) and Volden et al. (2001) observed that less than 5% of the total duodenal AA flow was contributed by passage of ruminal FAA. These studies indicate that when taking into account all soluble N-fractions, approximately 8% of the dietary soluble N will escape the rumen and, thus, contribute to the intestinal amino acid flow and thereby affect the protein values of the feed.
The in situ method is one of the techniques that have achieved widest application for measuring ruminal CP degradability, and it is used as the reference method in several protein evaluation systems for ruminants. In these systems effective degradability is calculated according to the following equation: EDP1 = a + (b x c)/(c + kpi), where a, b, and c are as described earlier, and kpi is the ruminal passage rate of b. This model assumes that the soluble "a fraction" is degraded at infinitive rate and that only insoluble feed N (b fraction) can escape the rumen. However, if we take into account that parts of the soluble N can flow out of the rumen with the liquid fraction, a two-compartment model can be used to estimate ruminal CP degradability. This model is expressed by EDP2 = (a x cs)/(cs + kps) + (b x c)/(c + kpi), where a, c, b, and kpi are as described earlier, and cs and kps are the ruminal degradation rate and passage rate of the soluble CP fraction out of the rumen, respectively. Ruminal escaped CP (REP) can be estimated as follows: REPi = 100 - EDPi (i=1,2). Based on the in situ results in Table 2, average degradation rate of soluble N in Table 5, and a fractional passage rate for soluble and insoluble fractions of 0.16 and 0.04, the EDPi=1,2 for GN1 and SN3 can be computed to:
 |
 |
 |
and
For GN1 the estimated ruminal escape of CP from REP2 model was only 4% greater than that computed from the REP1 model. However, for SN3 the effect was bigger and the ruminal escape was 32% greater when estimated from the REP2 model than from the REP1.
No difference was observed in degradation rate and ruminal escape between the fresh grass (GN1) and the corresponding grass silage (SN1, Table 6). The same effect has also been observed in vitro for alfalfa (Makoni et al., 1994). Nevertheless, for the GN1, only 24% of the N dose was converted to ammonia, whereas for the silage it was three times higher. For all silages, across dose size, on average 76% of the N dose was converted to NH3. In a tracer study using 15N, Siddons et al. (1985) examined the N metabolism in the rumen of sheep fed grass silage. They estimated that 69% of the ingested N was converted to NH3. Using the same approach in sheep fed either fresh white clover or perennial rye grass, Nolan (1993) reported that on average 60% of the ingested N was degraded through the rumen ammonia pool. However, in these studies calculations included total ingested N and not only soluble N, which may give lower values than those observed for grass silage extracts in the present study. In an experiment with cattle given casein, Mangan (1972) found that 43% of the casein was converted to NH3. The studies sited above report a higher conversion factor than observed for the fresh grass in the present study. Wallace (1996) emphasized that if energy is insufficiently supplied or when the rate of peptide degradation exceeds the rate of AA used for microbial protein synthesis, peptide catabolism leads to excessive NH3 production. This means that low conversion efficiency in grass silage could be explained by an imbalance of dietary N and energy. In a study with dry cows fed grass silage (Rooke et al., 1987), intraruminal infusion of easily fermentable glucose gave an increased microbial N synthesis, which partly could be explained by a better synchronization of rapidly degradable N and carbohydrates. In the present study, the soluble extract of GN1 had a lower N concentration and probably a higher content of easily fermentable carbohydrates than the silage extracts. In silage, water-soluble carbohydrates have been replaced by fermentation products, which are unable to supply sufficient energy to support microbial growth. This could possibly explain why a lower proportion of the ruminally administered N in GN1 was converted to ammonia than in the silages. These results show that it is important to feed adequate amounts of easily fermentable carbohydrates when grass silage is the main forage in the diet.
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Implications
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Results obtained in this study demonstrate that soluble dietary N fractions are not completely degraded in the rumen and, therefore, contribute to the duodenal AA flow. One way to improve the measurements of the protein value of grass silage is to include a two-compartment model when estimating in situ protein degradability. Furthermore, this study shows that if the main objective is to predict ruminal escape of soluble N, a simple model can be developed for prediction. However, if the main objective is to study the degradation kinetics of the individual soluble N-fractions, a complex model should be developed. The complex model could also be used to study synchronization of N and energy for microbial growth, which may improve the N utilization in ruminants. Results obtained in this study indicate that when grass silage is the main forage it is important to have sufficient amount of easily fermentable carbohydrates in the diet.
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Footnotes
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1 We gratefully acknowledge K. Hove for performing surgery on the experimental animals, M. Henne for technical assistance, and H. Steinshamn for providing the experimental forage. 
Received for publication December 6, 2001.
Accepted for publication May 7, 2002.
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Top
Abstract
Introduction
proteins and long-chain peptides; 
short-chain peptides; • free amino acids; 
ammonia. Mean values of three cows. Vertical bars describe SE.