J. Anim Sci. 2008. 86:1364-1371. doi:10.2527/jas.2007-0736
© 2008 American Society of Animal Science
Evaluation of the fermentation dynamics of soluble crude protein from three protein sources in continuous culture fermenters1
A. Bach*,
,2,
M. Ruiz Moreno
,
M. Thrune and
M. D. Stern
* Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain;
and
Grup de Recerca en Nutrició, Maneig, i Benestar Animal, Unitat de Remugants, Institut de Recerca i Tecnologia Agroalimentàries, 08193 Bellaterra, Spain; and
Department of Animal Science, University of Minnesota, St. Paul 55108
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Abstract
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Eight dual-flow continuous culture fermenters (1.03 ± 0.05 L) were used to assess differences in microbial degradation of the soluble CP fraction of canola meal (CMSCP), soybean meal (SBMSCP), and fish meal (FMSCP) using a completely randomized design with two 9-d experimental periods and a solution of tryptone as a control treatment (control). All fermenters received the same basal diet (58% ground corn, 40% canary grass hay, 0.4% vitamin-mineral premix, 1% CaCO3, 0.6% salt on a DM basis) in 8 equal portions daily. During sampling on the last 3 d of each period, 90-mL doses containing soluble CP were infused into the fermenters 30 min after the beginning of the first and last feedings of the day. The total amount of soluble CP supplied by the infusions of FMSCP, CMSCP, and SBMSCP was 3.2 g/d, representing 24% of the daily dietary CP intake. Infusion of FMSCP resulted in the greatest (P < 0.05) NH3-N concentration (4.6 ± 0.40 mg/dL) compared with the other treatments (0.5 ± 0.40 mg/dL). Microbial N flow (g/d) from the fermenters was also greatest (P < 0.05) with FMSCP (1.42 ± 0.062) compared with the other soluble CP fractions (1.08 ± 0.062). The efficiency of microbial protein synthesis tended to be lowest with the control diet, and the efficiency of N utilization was lowest with FMSCP treatment. These results indicate that N was limiting microbial growth in the control diet, and there was more rumen-available N with the FMSCP diet compared with the other dietary treatments. The extent of degradation of the soluble CP fraction from fish meal, soybean meal, and canola meal was determined to be 99, 30, and 37% of soluble CP, respectively. These results indicate that the soluble CP fraction is not 100% degraded in all feeds and that assuming a high degradation extent of the soluble CP fraction from soybean meal and canola meal may result in an underestimation of the supply of undegradable protein from these protein sources.
Key Words: in vitro • protein degradation • soluble protein
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INTRODUCTION
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Accurate estimation of rate and extent of ruminal protein degradation is a prerequisite to accurate ration formulation, particularly for high-performing animals in which microbial protein cannot meet requirements for MP and must be supplemented with feed protein escaping rumen degradation. Protein degradation is often estimated using the in situ technique. Apart from problems associated with this method, such as particle loss, microbial contamination, and low reproducibility (Hvelplund and Weisbjerg, 2000
), the in situ method cannot estimate degradation of soluble CP.
Chaudhry and Webster (2001) used a gel electrophoresis technique to assess ruminal degradation of soluble CP of several feeds; resistance to, or escape from, rumen degradation of soluble CP varied with class of feed and with type and molecular weight of soluble CP. Schwingel and Bates (1996) used electrophoresis and showed that approximately 30% of soluble CP from soybean meal (SBM) incubated with mixed ruminal micro-organisms was not degraded after 9 h. Hedqvist and Udén (2006) measured degradation of soluble CP of 20 feedstuffs in vivo and in vitro and found drastic differences in degradation of soluble CP among protein sources. These results indicate that a fraction of soluble dietary CP might not be ruminally degraded and can, therefore, supply the animal with amino acids. However, most dairy feeding models (INRA, 1989; NRC, 2001) assume that all soluble CP is instantaneously degraded in the rumen.
Our main objective was to assess differences in the microbial degradation of soluble CP fraction of 3 common protein sources and associated repercussions on microbial growth. The protein sources selected were canola meal (CM) as an ingredient rich in degradable protein and relatively low in CP solubility; SBM as an ingredient with highly degradable protein and relatively high CP solubility; and fish meal (FM) as an ingredient containing poorly degraded protein and relatively low CP solubility.
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MATERIALS AND METHODS
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All procedures were approved by the Animal Care Committee of the University of Minnesota.
Diets and Protein Sources
A basal ration consisting of 58% ground corn, 40% canary grass hay, 0.4% vitamin-mineral premix, 1% calcium carbonate, and 0.6% salt, on a DM basis, was prepared with the nutrient composition depicted in Table 1. To ensure that CP was the limiting factor for microbial growth, the diet was designed to provide a high supply of nonfibrous carbohydrates (NFC) relative to CP (an NFC:CP ratio greater than 3). All ingredients of the basal diet were ground through a 2-mm screen and pelleted with a pellet mill (CL-5, California Pellet Mill Co., Crawfordsville, IN) to a final dimension of 6 mm in diameter x 10 mm long.
Samples of FM (69.8% CP on a DM basis; 22% soluble CP as a percentage of CP), CM (40.6% CP on a DM basis; 21.1% soluble CP as a percentage of CP), and SBM (49.9% CP on a DM basis; 35.9% soluble CP as a percentage of CP) were ground to 2 mm and then soaked in distilled water (1:4, wt/vol) at 38° C for 1 h under continuous stirring. Samples were then centrifuged at 1,000 x g for 10 min, and the supernatant was filtered through a N-free filter paper (25-µm pore size) under vacuum. Filtrates were collected, and their N content was determined. Because not all soluble CP filtrates had the same CP concentration, they were normalized to a final CP concentration of 1.8% as fed, which corresponded to the lowest CP content of the 3 filtrates. This normalization was accomplished by diluting the 2 filtrates with the greatest CP concentration with distilled water to reach the same CP concentration as the filtrate with the lowest CP content (1.8% CP as fed).
Normalized filtrates were stored at –20° C in 90-mL doses and gently thawed at room temperature with a fan for approximately 20 min before infusion into the fermenters. A fourth solution of tryptone (BP1421–500, Fisher Bioreagents, Fairlawn, NY) was prepared with 3.25 g of tryptone (100% CP as a percentage of DM and 99.1% CP solubility as a percentage of total CP) per liter. This solution was also stored at –20° C in doses of 90 mL and was used as a negative control during the infusions. The CP content of the tryptone solution was equivalent to the CP content of the artificial saliva (Weller and Pilgrim, 1974) infused in the fermenters but supplied amino acids instead of nonamino acidic N. There were a total of 4 treatments: the basal diet plus a twice-a-day 90-mL infusion of the tryptone solution (control), the basal diet plus a twice-a-day 90-mL infusion of the SBM solution (SBMSCP), the basal diet plus a twice-a-day 90-mL infusion of the FM solution (FMSCP), and the basal diet plus a twice-a-day 90-mL infusion of the CM solution (CMSCP).
Continuous Culture System Operation
Eight dual-flow continuous culture fermenters (1.03 ± 0.05 L), as described by Hannah et al. (1986), were inoculated with ruminal fluid obtained from a cannulated dry cow fed a 70:30 forage:concentrate diet (containing 11% alfalfa hay, 58% corn silage, 4.2% wheat straw, 8.4% ground corn, 5.4% dried molasses, 4.4% SBM, 5.7% soybean hulls, 0.5% blood meal, and 2.4% vitamin premix; DM basis). The fermenters were fed a mean of 75 ± 0.3 g/d of pelleted dietary DM by an automated feeding device (Hannah et al., 1986) that was operated under the control of an automatic electronic timer (DT 17, Intermatic, Spring Groove, IL). Fermenters received 8 equal portions of the total daily DM fed every 3 h. Each feeding phase had a duration of 1.5 h. Feeding times and duration of feedings were simultaneous for all fermenters and controlled by an electronic timer. Solids and liquid passage rates were adjusted daily to 5.5 and 10%/h, respectively, by regulating buffer input and filtrate removal rates. Culture pH was automatically maintained at a minimum of 6.0 by an automatic addition of 5 N NaOH when necessary. The experiment was conducted in two 9-d periods, with 6 d for adaptation and stabilization of the fermenters followed by 3 d for sampling. Treatments were randomly assigned to each fermenter within period.
During the sampling days, 30 min (0900 h) after the beginning of the first morning feeding (0830 to 1000 h feeding cycle) and 30 min (2100 h) after the beginning of the evening feeding (2030 to 2200 h feeding cycle), the fermenters received a 90-mL infusion containing the isonitrogenous, soluble CP fractions from FM, CM, SBM, or tryptone, depending on the treatment, at a rate of 3 mL/min with a screw-driven, syringe, constant-infusion pump (Harvard Apparatus Co., Holliston, MA). Five minutes before and after each infusion, artificial saliva flow (approximately 1.73 mL/min) to the fermenters was interrupted to compensate for the increased output flow rate during infusions. The total amount of soluble CP supplied by infusion of FMSCP, CMSCP, and SBMSCP was 3.2 g/d. The basal ration CP concentration was 11.7% on a DM basis, and this figure was brought to 15.3% CP (DM basis) after the infusion of 3.2 g/d of soluble CP. Therefore, the infused soluble CP fractions accounted for 24% of the total dietary CP provided. The amount of CP supplied by tryptone (control) was 0.56 g/d (6% of the total dietary CP) and substituted for the amount of N that would have been supplied by artificial saliva during the infusion times. Infusions of soluble CP were conducted during the last 3 d of each period, not throughout the experiment, because the objective was to evaluate the degradation properties of the protein sources used, rather than assessing their effects on microbial fermentation.
Sample Collection and Analytical Procedures
During the 3-d sampling period, fermenter effluents were maintained in a 1° C water bath to retard microbial and enzymatic activities. Three separate 500-mL samples from each fermenter effluent were taken and homogenized during the last 3 d of each period using a homogenizer (PT10/3S, Kinematica GmbH, Bohemia, NY), stored at –20° C, and composited within fermenter at the end of the study. Then, a subsample (approximately 500 mL) of the composite effluent samples was lyophilized (25SL, Virtis, Gardiner, NY) and used for DM, OM, NDF, ether extract, ash, and bacterial purine determinations. Frozen composite samples were used for total N and NH3-N. On each sampling day, 0, 0.5, 1, 3, and 6 h after the morning infusion of soluble CP fractions, a 10-mL aliquot from each fermenter flask was obtained through the filtrate lines. Five milliliters of this sample was preserved with 0.2 mL of 0.2 N sulfuric acid and was stored at –20° C for subsequent NH3-N analysis, and the remaining 5 mL was preserved with 1 mL of m-phosphoric acid (250 g/L) and also was stored at –20° C until subsequent VFA analysis.
At the end of each experimental period, the fermenter contents were strained through 2 layers of cheesecloth and centrifuged at 1,000 x g for 10 min to remove feed particles and eukaryotic microorganisms. The supernatant was centrifuged at 20,000 x g for 20 min to remove bacteria. Bacterial pellets were resuspended in distilled water, frozen, and lyophilized. The VFA concentrations of the fermenter fluid were analyzed using a GLC (model 5880A, Hewlett Packard, Palo Alto, CA) with a Carbopack DA/0.3% Carbowax 20M column (Supelco, Bellefonte, PA). Neutral detergent fiber concentrations of the diets and fermenter effluents were determined using the procedure of Van Soest et al. (1991). Ash content of the diets and fermenter effluents was determined after a 24-h combustion at 550° C (AOAC, 1984). Ether extract of the diets and fermenter effluents was determined by ethyl ether extraction (AOAC, 1984). The concentration of NFC was estimated by subtraction of ether extract, NDF, and CP concentrations from the sample OM. Determination of NH3-N concentration in the fermenter fluid was conducted by steam distillation using a Kjeltech 1030 AutoAnalyzer (Tecator, Herndon, VA). The rest of the N determinations (fermenter effluent, microbial, dietary, soluble CP solutions, etc.) were conducted using the macro-Kjeldahl (AOAC, 1984) procedure. The CP solubility of the ingredients used in the diets was determined by filtration (Crooker et al., 1978). Purine concentrations in individual bacterial samples and fermenter effluents were used to separate N into bacterial and dietary fractions (Zinn and Owens, 1986).
Calculations and Statistical Analysis
Crude protein degradability of the basal diet was calculated by subtracting the amount of soluble CP supplied by tryptone (assuming that it was 100% ruminally degradable) from the measured total CP degradation in the fermenters. Even if the degradation of soluble CP from tryptone was not 100%, the error made would be low (less than 6%, because tryptone accounted for 6% of the total dietary CP). Degradability of the soluble fraction of CP from FM, SBM, and CM was estimated by subtracting 76% from the CP degradability (proportion of dietary CP supplied by the basal ration) of the control diet (excluding the degradable fraction provided by tryptone, as described above) from the observed total CP degradation of FMSCP, SBMSCP, and CMSCP, respectively, in the fermenters and transforming the result to a 100 scale by dividing by 0.24 (the proportion of protein in each diet that was supplied by soluble CP from FM, SBM, and CM). The efficiency of microbial protein synthesis (EMPS) was calculated as grams of microbial N per kilogram of OM truly fermented and was considered an estimate of the efficiency of energy utilization by rumen bacteria. The efficiency of N utilization (ENU) by rumen bacteria was calculated as grams of microbial N divided by grams of available N (calculated as N intake, including urea from artificial saliva and soluble CP infusions, minus undegraded N). Digestion of DM, OM, NDF, and CP, and flows of total N, nonammonia N, microbial N, and dietary N, were calculated as described by Stern and Hoover (1990).
Data obtained sequentially (VFA and NH3-N concentrations) around fermenter infusions were analyzed as a randomized complete design with repeated measures, using the following mixed-effects model:
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where Fermenter and Period entered the model as random effects, Treatment represented the fixed effect of the soluble CP fraction being infused, Time was considered a repeated factor, and the interaction of Fermenter with Period nested within the interaction between Treatment and Time (the error term) was subjected to 3 variance-covariance structures including compound symmetry, unstructured, and autoregressive order 1. The variance-covariance matrix that yielded the smallest Schwarz’s Bayesian criterion was considered to be the most desirable structure (Littell et al., 1996). The Student-Neuman-Keuls’ test (Montgomery, 1996) was used for mean comparisons among diets. Data corresponding to bacterial growth, EMPS, ENU, NH3-N, and VFA concentrations, and OM, CP, NFC, and NDF degradabilities, were analyzed with the same model described above, with repeated measures (time) and random effect of fermenter omitted. The significance level for the overall model was set to P 
0.05, and tendencies were declared at P 0.10. Differences among treatment means were only explored when the significance of the overall model was P 0.05. All statistical analyses were conducted using SAS (SAS Inst. Inc., Cary, NC).
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RESULTS AND DISCUSSION
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True OM digestibility (Table 2) tended (P = 0.08) to be different among treatments, with the lowest values observed with the SBMSCP and CMSCP treatments and the greatest values with the control and FMSCP treatments. The relatively low true OM digestibility observed with SBMSCP was possibly the consequence of low NDF and NFC digestion (Table 2). A similar value for NDF digestion was observed with FMSCP, but in this case, a high CP degradation (Table 2) counterbalanced the low NDF digestion and tended to result in a high true OM digestion. The low OM digestibility obtained with CMSCP was probably due to the relatively low NFC digestion observed with this treatment (Table 2). Degradation of CP tended (P = 0.08) to differ among treatments, with the greatest value corresponding to FMSCP. This result was unexpected, because FMSCP was included in the study as a soluble CP from a slowly degradable protein (NRC, 2001). This high CP degradation was due to the high degradability of the soluble CP from FM compared with those of SBM and CM as discussed later.
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Table 2. Effects of supplemental protein source solubility on digestion and N metabolism in continuous culture fermenters
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Ammonia-N concentrations were extremely low with the exception of those obtained with the FMSCP treatment. Despite a greater (P < 0.05) NH3-N concentration with the FMSCP treatment, NH3-N did not attain the 5 mg/dL concentration usually recommended to ensure maximum microbial growth (Satter and Slyter, 1974). However, Russell et al. (1983) reported no difference in microbial growth when NH3-N concentrations were below 5 mg/dL or greater than 16 mg/dL. Owens and Bergen (1983) summarized several studies and concluded that the minimum amount of NH3-N to maximize bacterial growth was 2.5 mg/dL. Other studies have also reported NH3-N concentrations below 5 mg/dL without reporting differences in microbial yield (Russell et al., 1983; Bach et al., 1999). The low NH3-N concentration in the current study was partially due to the experimental design and dietary treatments. It is possible that total microbial growth could have been limited by these low NH3-N concentrations, but the objective was to assess the extent of utilization of the soluble CP fractions by ruminal bacteria maintained in an environment in which energy would not limit growth. However, bacteria composition was not affected by treatments, and it was in range to that previously reported in the literature (Bach et al., 1999) with an average of 6.5 ± 0.24% N and 80.8 ± 0.02% OM on a DM basis. Because the diets used in the current study were rich in NFC, the maximum degradation extent of soluble CP should have been reached, because microbes that ferment NFC use not only NH3 but also peptides and amino acid as N sources, whereas the microbes that ferment NDF use NH3-N as their main N source (Russell et al., 1992). Jones et al. (1998) concluded that with diets rich in NFC (such as the ones used in the current study), the reduction in NH3-N concentration in the rumen may limit the growth of fiber-digesting microbes. Thus, it is likely that NDF degradation in the current study was impaired (as indicated by the relatively low NDF digestibilities observed), but this fact should have not limited the degradation extent of the soluble CP fraction. Furthermore, true OM degradation was within a range, or above, values previously reported in the literature (Bach et al., 1999; Ariza et al., 2001; Griswold et al., 2003; Lean et al., 2005). This would indicate that the low NH3-N concentrations found in the current study did not prevent bacteria from thoroughly fermenting OM.
As expected, total N flow from the fermenters was lowest (P < 0.05) with control compared with the remaining treatments, because control fermenters received the lowest amount of N. Ammonia-N flow from the fermenters was greatest (P < 0.05) with FMSCP as a consequence of the greatest NH3-N concentration observed with this diet (Table 2
). Flow of nonammonia-N was greatest (P < 0.05) with SBMSCP and CMSCP, intermediate with FMSCP, and lowest (P < 0.05) with control. These differences were due to the fact that both SBMSCP and CMSCP resulted in the greatest (P < 0.05) flow of dietary N due to the tendency observed for these diets to have a low total CP degradation (Table 2). Conversely, FMSCP resulted in the greatest (P < 0.05) microbial N flow, followed by CMSCP and SBMSCP and control. This difference is related to the tendency observed in CP degradation, which was greatest in FMSCP, and thus it provided the greatest amount of available N to sustain microbial growth. The fact that control had the lowest microbial N flow was due to the lower CP supplied by this diet.
The EMPS observed in the current study is within the range previously reported in the literature (Illg et al., 1994; Bach et al., 1999). There was a tendency (P = 0.09) for the EMPS to be different among treatments (Table 2), with the control diet resulting in the lowest EMPS, which was probably due to the lower N supply of this diet. In contrast, the ENU was close to 100% with the control diet. The low EMPS and high ENU are a clear indication that N was limiting microbial growth in the control diet as might have been expected due to low CP and high NFC contents of this treatment. The control treatment supplied the same amount of N that would have been supplied by artificial saliva during the infusion times but in the form of amino acid and peptides instead of urea to mimic the supply of soluble CP from FMSCP, SBMSCP, and CMSCP. Bacterial growth has been shown to increase with addition of amino acid or peptides in cellulolytic and amylolytic bacteria (Kernick, 1991; Atasoglu et al., 2001). In contrast, the FMSCP diet resulted in the lowest (P < 0.05) ENU, which further supports the observation that there was more microbial-available N with the FMSCP diet compared with the other dietary treatments, because a greater proportion of the available CP was actually not utilized to sustain microbial protein synthesis compared with the rest of the treatments. Overall, values for ENU in the current study are much greater than those previously reported (Bach et al., 1999; Bach and Stern, 1999; Ariza et al., 2001). High ENU values found in the current study are probably due to the high NFC:rumen-available CP ratio that the diets provided. In this study, NFC:rumen-available CP ranged from 7.2 in the control treatment to 4.3 with the FMSCP treatment. Under these conditions, it is reasonable to expect that because energy supply was abundant relative to protein supply, microbes used no or little quantities of protein as energy source, which would explain the high ENU values observed.
Total production and molar proportions of VFA were not affected by treatment (Table 3). Consistent with this observation, no differences were found in VFA molar proportion at 0.5, 1, 3, and 6 h after infusion of the soluble CP fractions of FM, CM, SBM, or tryptone (data not shown). A difference in molar proportions of branched-chain VFA (BCVFA) might have been expected because of differences in CP degradation among treatments, especially with the FMSCP diet. Branched-chain VFA are produced by rumen microbes when degrading protein (Bryant, 1973). Despite no differences in BCVFA molar proportions among treatments (Table 3), the FMSCP treatment had numerically greater BCVFA concentrations. These numerically greater BCVFA concentrations could also be linked to the lower ENU and greater CP degradation observed with FMSCP compared with the other treatments.
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Table 3. Effects of supplemental protein source solubility on total VFA production and molar proportions in a continuous culture
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The experimental design used in this study allowed the determination of the degradation extent of the soluble CP fraction of SBM, FM, and CM. Table 4 shows that the degradability of the soluble CP of FM was greatest (P < 0.05) and was approximately 3-fold that of CM or SBM. In agreement with this observation, NH3-N concentration in the fermenters flasks after infusion of the soluble CP fractions was consistently greatest (P < 0.05) at each time point with FMSCP compared with CMSCP and SBMSCP (Figure 1). This result was surprising, because FM is considered a source of rumen-undegradable protein. The current study illustrates that almost 100% (Table 4) of the soluble CP from FM is degradable. However, it is interesting to note that only 30 and 37% of the soluble CP from SBM and CM, respectively, is degraded. Therefore, the degradability of the soluble CP fraction from different protein sources does not seem to be positively correlated with the degradation extent of the insoluble CP fraction of each protein source. The degradability values for the soluble CP fraction of CM are relatively close to those previously reported by Hedqvist and Udén (2006). These authors reported an effective degradation of the soluble CP fraction of 44% of total soluble CP for CM. However, Hedqvist and Udén (2006) reported an effective degradation of the soluble CP fraction of 74% of total soluble CP for SBM, which is much greater than the 30% found in the current study. These differences may be attributed to the methods used. Hedqvist and Udén (2006) used an in vitro batch culture modified from that described by Broderick (1987), whereas the values from the current study were derived from continuous culture. Also, Hedqvist and Udén (2006) did not provide the same amount of CP into the incubation batches, and the ratio of available CP to available energy was also different.
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Table 4. Estimated degradability values for the soluble CP fraction of soybean meal (SBM), fish meal (FM), and canola meal (CM)
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The soluble CP fraction of FM is extensively degraded, but that of SBM and CM, 2 protein sources with a relatively high content on degradable CP, seems to be poorly degraded. Current nutritional models, such as the NRC (2001), estimate total degradable CP by assuming a 100% extent of degradation for the soluble CP fraction. Based on the results of the current study, this assumption seems to be valid for FM but nor for CM or SBM. Under the assumption that soluble CP is 100% ruminally degraded and using degradation rates from the NRC (2001) and a passage rate of 6%/h, the rumen-degraded protein values for FM, CM, and SBM would be 35.7, 57.4, and 69.4% of CP, respectively. However, if the degradability values for soluble CP obtained in this study are used, keeping the rest of the parameters as described above, the rumen-degraded protein values for FM, CM, and SBM would be as follows: 35.4, 44.1, and 53.5%. Therefore, it could be concluded that if the degradability of the soluble CP fraction is assumed to be 100%, there is a risk of underestimating rumen-undegradable protein values of some CP sources.
In conclusion, the soluble CP fraction of some CP sources may be far from 100% degradable in the rumen. Results from this study suggest that current feeding systems underestimate the undegradable protein values of major protein sources such as SBM and CM.
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Footnotes
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1 We would like to thank Mary Raeth-Knight (University of Minnesota) for her valuable assistance in conducting VFA analyses; Cargill (Elk River, MN) and Soybest (West Point, NE) for providing the samples of canola meal, fish meal, and soybean meal, respectively; and Hans Jung (University of Minnesota) for providing the canary grass. Also, the Ministerio de Educación y Ciencia of the government of Spain is thanked for funding the living and travel expenses of Alex Bach to conduct this study through the Programa de Movilidad PR2006-0146. 
2 Corresponding author: alex.bach{at}irta.es
Received for publication November 16, 2007.
Accepted for publication February 26, 2008.
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Abstract
INTRODUCTION
= soybean meal; 
= canola meal. *Indicates a significant difference (P < 0.001) between fish meal and the rest of treatments at each time point.