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Rumen escape protein in grass and grass silage determined with a nylon bag and an enzymatic technique
Animal Feed Science and Technology 111 (2004) 1–9
Rumen escape protein in grass and grass silage determined with a nylon bag and an enzymatic technique J.W. Cone∗ , A.H. van Gelder, A.A. Mathijssen-Kamman, V.A. Hindle Nutrition and Food, Animal Sciences Group of Wageningen UR, P.O. Box 65, NL-8200 AB Lelystad, The Netherlands Received 27 February 2003; received in revised form 18 June 2003; accepted 5 August 2003
Abstract Rumen escape protein (REP) was determined for six grasses and 16 grass silages using a nylon bag technique and an in vitro technique using a proteolytic enzyme preparation of Streptomyces griseus. In vitro, the samples were incubated for 0, 1, 6 and 24 h. The highest correlation observed between percentage REP (%REP), determined with the nylon bag technique, and the in vitro undegradable fraction (U) occurred at 24 h of incubation (%U24; R2 = 0.71). Shorter incubation periods weakened the relationship (R2 = 0.46–0.59). Percentage REP could be estimated from %U24 as %REP = 3.3 (±4.02) + 1.31 (±0.19) ∗ %U24. For the incubation periods investigated, results show that the highest relationship with %REP occurred with an incubation period of 24 h for these grass and grass silage samples. © 2003 Elsevier B.V. All rights reserved. Keywords: Nylon bag technique; Protease; Rumen escape protein; Streptomyces griseus
1. Introduction Many protein evaluation systems for ruminants, such as the Dutch DVE/OEB system (Tamminga et al., 1994) require data obtained from a nylon bag technique. Nylon bag techniques divide feed protein into a washout fraction (W), a rumen undegradable fraction Abbreviations: CP, crude protein (6.25 ∗ N); D, potentially rumen degradable fraction; DM, dry matter; kd , rumen degradation rate in % h−1 ; N, nitrogen; NDF, neutral detergent fibre; REP, rumen escape protein; U, undegradable fraction; %U, percentage in vitro undegradable N; W, washout fraction ∗ Corresponding author. Tel.: +31-320-237354; fax: +31-320-237320. E-mail address: [email protected] (J.W. Cone). 0377-8401/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2003.08.004
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(U) and a potentially rumen degradable insoluble fraction (D = 100 − [W + U]). The degradation rate (kd ) of D can be calculated from the rate of disappearance of nitrogen (N) from the nylon bags (Robinson et al., 1986). Assuming a ruminal passage rate, the percentage rumen escape protein (%REP) can be calculated by the equation of Ørskov and McDonald (1979). An enzymatic technique, using protease from Streptomyces griseus was described by Krishnamoorthy et al. (1983) as a laboratory technique to determine %REP, which is suitable for rapid screening of samples. The technique was further developed by Aufrère et al. (1991) determining N hydrolysis after 1 and 24 h incubation at pH 8.0, and using correction factors for groups of feedstuffs. Cone et al. (1996) simplified this method using the undegradable fraction after 24 h of incubation without correction factors. Cone et al. (1996) used samples of concentrates, concentrate ingredients and forages. However, in a later study, Cone et al. (2002) showed that for concentrate ingredients, the highest correlation was observed between %REP determined with a nylon bag technique and the in vitro undegradable fraction after 1 h of incubation. This raised the question of which in vitro incubation period has the highest relationship with %REP, based on a nylon bag technique for grass and grass silage samples. 2. Materials and methods 2.1. Feed samples and chemical analysis Grass samples were harvested after growth of 3 weeks on different dates in 1996 and 1997 (Table 1). Grass, predominantly Lolium perenne, was grown on a clay soil (Lelystad, The Netherlands) and on a sandy soil (Cranendonck, The Netherlands). Pastures were fertilised with 150 or 300 kg N/ha per year. Harvested samples were stored at −20 ◦ C for later nylon bag incubations or freeze dried and ground to pass a 1 mm screen for chemical analysis and in vitro incubations with protease of S. griseus. At harvest, sub-samples of grass were wilted at 25 or 45% dry matter (DM) and ensiled in 30 l plastic containers. After 8 weeks at 17 (±1) ◦ C, containers were stored at 3 ◦ C until use. Sub-samples from the silages were used for nylon bag incubations, while other sub-samples were freeze dried and ground (1 mm) for chemical and in vitro analysis. Dry matter content was determined after 4 h at 103 ◦ C (ISO 6496) and ash after 3 h at 550 ◦ C (ISO 5984). Crude protein (CP) was determined using a Kjeldahl method (ISO 5983). Sugars were measured colorimetrically, as described by Van Vuuren et al. (1993), after extraction with 40% ethanol followed by treatment with 0.023 M HCl to hydrolyse the bonds between sugar molecules. Neutral detergent fibre (NDF) was determined by a modified method of Van Soest et al. (1991), without use of sodium sulphite. After boiling with ND, the residue was incubated in 60 ml Na–K–phosphate buffer (pH 7.0) for 15 min at 40 ◦ C with 15 mg amylase (Thermamyl 120, NOVO Nordisk, Bagsvaerd, Denmark) and 0.25 ml protease (Alcalase 2.4 l, NOVO Nordisk, Bagsvaerd, Denmark) to remove residues of starch and protein. Lipids were then removed by washing with acetone until the acetone was colourless. NDF is expressed without residual ash.
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Table 1 Chemical compositiona (g/kg DM) of the grasses and grass silagesb Soil type
Year
N-fertilization (kg/ha per year)
Harvest date
DM (g/kg)
Ash (g/kg DM)
CP (g/kg DM)
NDF (g/kg DM)
Sugars (g/kg DM)
Grass Clay
1996
150 150 300 300
17 June 18 July 3 June 9 September
227 206 187 168
79 99 85 100
138 169 241 269
513 401 417 436
172 204 137 62
Sand
1997
300 300
17 June 30 June
163 169
103 88
292 156
399 431
78 226
187
92
211
433
146
1997
Mean grass Silage ± 25% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
237 275 227 331 237
93 110 96 106 111
152 190 245 157 281
500 401 412 423 381
30 63 18 109 17
150 300 300
4 August 17 June 30 June
294 261 288
90 106 112
128 300 213
524 358 484
108 51 18
269
103
208
435
52
Mean silage 25% Silage ± 45% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
446 509 431 487 455
89 111 95 105 108
152 192 243 156 275
514 419 429 425 401
93 115 48 133 32
150 300 300
4 August 17 June 30 June
489 502 571
87 109 102
132 305 213
544 391 498
101 77 66
486
101 1.0
209 1.6
453 3.0
83 3.1
Mean silage 45% S.E.M. a b
All samples analysed in duplicate. Pastures were fertilised with 150 or 300 kg N/ha per year.
2.2. In situ nylon bag measurements Grass and silage samples were thawed and cut with a paper guillotine to lengths of approximately 3 cm. CP degradation was determined with the nylon bag technique of Ørskov and McDonald (1979) as modified by Van Vuuren et al. (1989). The W fraction was defined as the fraction disappearing by washing with cold tap water in a washing machine for 45 min.
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Disappearance of N from feed samples (5 g DM) in nylon bags was determined after 3, 6, 12, 24, 48 and 336 h of rumen incubation in triplicate in each of three cows (Van Vuuren et al., 1989). The cows received daily two meals of 8 kg DM, composed of 35% grass silage, 35% maize silage and 30% concentrate. The U fraction was determined as the residual fraction after 336 h of incubation in the rumen. W and U were used as fixed values and the residues were fitted to a first order degradation model (Robinson et al., 1986). Calculations were without a degradation lag time. The %REP was calculated from the nylon bag incubations, assuming a rumen passage rate of 4.5% h−1 (Vérité et al., 1987) and the degradable (D) fraction to be 100 − [W + U]. In the silage samples, 5% of the W fraction was regarded as rumen escape protein according to the Dutch DVE/OEB system (Tamminga et al., 1994). 2.3. In vitro measurements using protease Soluble N was measured after 0, 1, 6 and 24 h of incubation with enzymes in borate/phosphate buffer at pH 8.0 as described by Aufrère and Cartailler (1988) and Cone et al. (1996). The protease from S. griseus (type XIV, Sigma P-5147, St. Louis, MO, USA) was used in a concentration of 20 mg in 1 l buffer. Tetracyclin (1 mg/l, Sigma N-3503) and nystatin (10 mg/l, Sigma T-3258) were added to the buffer to prevent microbial growth. Based on the N content, and soluble N, the percentage of undegradable N (%U) was calculated for the indicated incubation periods (%U0, %U1, %U6 and %U24). 3. Results 3.1. Chemical analysis Ash content ranged from 79 to 112 g/kg DM, CP from 132 to 305 g/kg DM, NDF from 358 to 544 g/kg DM and sugars from 17 to 226 g/kg DM (Table 1). There was no marked influence of ensiling on the chemical composition, with the exception of decreased sugar content in the silages. 3.2. Nylon bag determination of %REP The W fraction of protein ranged from 16% for a grass sample (sandy soil, 1997, 300 kg N/ha per year) to 66% for the silage counterpart (25% DM) of the same sample. U ranged from 7 to 8% for the fresh and silage samples harvested 17 June 1997 on the sandy soil (300 kg N) to 29 to 30% for the silage samples harvested on 4 August 1997 on the sandy soil (150 kg N; Table 2). Data from Table 2 were used to calculate %REP, which ranged from 16.8% to nearly 42%, and was on average lowest in the 25% DM silage samples (27.0%) and highest in the fresh samples (34.9%). 3.3. Enzymatic protein degradation The %U0 (insolubility of CP, Table 3) in the buffer with pH 8.0, without incubation with protease, was highest in fresh grass samples with an average of 58.2% and maximum 66.9%.
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Table 2 The washout (W) and undegradable (U) fractions and degradation rates (kd ) of CP determined with a nylon bag techniquea Soil type
Year
N-fertilizationb (kg/ha per year)
Harvest date
W (% of OM)
U (% of OM)
kd (% h−1 )
%REPc (% of CP)
1996
150 150 300 300
17 June 18 July 3 June 9 September
31 17 28 32
26 12 11 21
14.6 11.2 9.7 5.6
36.1 32.4 30.3 41.9
300 300
17 June 30 June
16 32
8 15
8.4 8.1
34.5 33.9
26.9
15.5
9.6
34.9
Grass Clay
1997 Sand
1997
Mean grass Silage ± 25% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
63 60 62 40 52
21 14 11 22 22
20.3 7.4 14.6 9.8 39.7
27.1 26.8 20.5 36.0 27.2
150 300 300
4 August 17 June 30 June
32 66 53
29 7 19
39.4 14.1 18.6
34.6 16.8 27.1
53.5
18.1
20.5
27.0
Mean silage 25% Silage ± 45% DM Clay
1996
1997 Sand
Mean silage 45% S.E.M.
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
53 55 50 23 38
20 17 12 22 21
11.2 8.0 12.5 15.0 10.1
30.4 29.8 24.6 35.8 35.5
150 300 300
4 August 17 June 30 June
27 41 39
30 8 17
14.0 8.9 17.9
41.8 27.2 27.8
40.8 2.2
18.4 2.3
12.2 2.60
31.6 –
a
All samples analysed in triplicate. Pastures were fertilised with 150 or 300 kg N/ha per year. c %REP was calculated from mean nylon bag parameters, using a passage rate of 4.5% h−1 . For silages 5% of W was assumed to be REP. b
The average %U0 was lowest in the 25% DM silage samples (30.8%), with a minimum of 23.0% (Table 3). The average %U0 of the 45% DM silage samples in the buffer with pH 8.0 was 37.6%. After 1 h in vitro, a considerable part (35.7%) of the insoluble CP in the fresh grass samples was degraded. Remaining insoluble CP after 1 h in vitro was, on average, 37.4%
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Table 3 The percentage undegraded protein (%U) after 0, 1, 6 and 24 h of in vitro incubation with a protease of Streptomyces griseusa Soil type
Year
N-fertilizationb (kg/ha per year)
Harvest date
%U0 (% of CP)
%U1 (% of CP)
%U6 (% of CP)
%U24 (% of CP)
1996
150 150 300 300
17 June 18 July 3 June 9 September
60.1 58.6 56.4 66.9
39.1 34.9 36.5 46.8
30.4 24.9 25.7 38.3
25.4 18.9 20.3 28.3
300 300
17 June 30 June
53.4 53.2
32.5 34.0
23.3 24.4
20.9 23.7
58.2
37.4
27.8
23.1
Grass Clay
1997 Sand
1997
Mean grass Silage ± 25% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
29.6 28.0 27.8 46.8 33.8
25.0 24.2 23.3 43.7 31.3
18.4 18.7 17.6 38.6 27.0
16.4 21.1 16.3 20.9 20.6
150 300 300
4 August 17 June 30 June
32.8 24.7 23.0
30.5 23.3 22.5
26.6 21.0 18.8
24.2 14.0 14.1
30.8
27.9
23.4
18.3
Mean silage 25% Silage ± 45% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
32.2 33.3 30.0 47.4 40.7
28.9 29.7 25.1 42.9 38.9
19.7 21.9 18.1 39.1 32.4
19.1 24.0 15.6 25.6 24.0
150 300 300
4 August 17 June 30 June
37.9 41.0 36.6
35.6 37.7 35.2
29.5 28.5 29.6
26.5 21.3 21.1
37.6 1.04
34.2 1.51
27.4 1.58
22.1 1.48
Mean silage 45% S.E.M. a b
All samples analysed in duplicate. Pastures were fertilised with 150 or 300 kg N/ha per year.
of total CP in the grass samples, ranging from 32.5 to 46.8%. After 1 h, CP degradation continued resulting in an average insoluble CP content of 23.1% after 24 h in vitro. After 24 h in vitro, on average, 60.3% of the insoluble CP (100 − %U0) was degraded in the grasses. The ensiled grass samples contained much less insoluble CP than fresh (non-ensiled) grasses. The %U0 was, on average, 30.8% for the 25% DM silages and 37.6% for the 45% DM silages. As a consequence, degradation of CP with Streptomyces proteases occurred more gradually in the silages than in the fresh grass samples. However, the %U24 was, on
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Table 4 Relationships between the nylon bag determined rumen escape protein (%REP) and percentage (%U) undegraded protein after 0, 1, 6 and 24 h of in vitro incubation with a Streptomyces griseus protease Incubation period (h) 0 1 6 24
Regression equation ∗ %U0
17.4 (± 3.40) + 0.33 (± 0.08) 8.9 (± 4.19) + 0.67 (± 0.12) ∗ %U1 14.2 (± 3.96) + 0.64 (± 0.15) ∗ %U6 3.3 (± 4.02) + 1.31 (± 0.19) ∗ %U24
R2
RSD
0.46 0.59 0.48 0.71
4.7 4.1 4.6 3.4
average, only slightly lower in the silages (18.3–22.1%) than in the fresh grass samples (23.1%). 3.4. Correlations between nylon bag and in vitro protein degradation Relationships between %REP determined in nylon bags (Table 2), and the in vitro determined %U after 0, 1, 6 and 24 h of incubation (Table 3), are in Table 4. %REP showed the highest correlation with %U after 24 h and the lowest after 0 h of incubation in vitro (i.e. solubility). Only the regression equation between %REP and %U24 had an intercept that did not differ from zero (Table 4 and Fig. 1). The slopes for all regression equations differed from one. Fig. 1 shows the relationships between the calculated %REP, determined with the nylon bag technique, and %U, obtained after 1 or 24 h incubation in vitro. From the figure, it is evident that the grass samples did not behave different from the silage samples. 4. Discussion Aufrère et al. (1991) estimated nylon bag determined %REP from in vitro enzymatic degradation after 1 and 24 h of incubation. Cone et al. (1996) showed that nylon bag estimated %REP was best correlated with %U24, using a mixed data set of mixed concentrates, concentrate ingredients and forages. Recently, Cone et al. (2002) showed that, for 26 concentrate ingredients, %REP was best estimated after only 1 h of incubation with S. griseus protease, suggesting that %REP in concentrate ingredients is mainly determined by its CP solubility. Licitra et al. (1998) showed for a number of feed samples a decreased residual-N by extending the incubation period from 8 to 18 h. Extending the incubation period from 18 to 24 or 30 h did not increase the solubility of N for the majority of the feed samples (Licitra et al., 1998). Licitra et al. (1999) showed that best regressions with in situ N degradation was obtained using a short incubation period (4 h) with high enzyme concentrations or prolonged incubation periods (36–48 h) with low enzyme concentrations. These results were confirmed by Mathis et al. (2001), investigating ruminal protein degradability of five forages. Our results show that for grass and grass silage samples incubated in nylon bags, %REP was best estimated by %U after 24 h of incubation in vitro, as the relationship between %REP and %U24 had the highest R2 , lowest RSD and an intercept that did not differ from zero. This suggests that for the investigated forage samples, %REP is determined partly by solubility and partly by degradation of CP by the S. griseus protease.
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Nylon bag technique estimated rumen escape protein (% of CP)
40
30
20
10 10
20
30
40
50
In vitro undegraded CP (% of CP) Fig. 1. Relationship between percentage rumen escape protein determined with a nylon bag technique and the in vitro percentage undegraded CP of grasses (, 䉱) and grass silages (䊊, 䊉) after 1 (䊊, ) or 24 (䊉, 䉱) h of incubation with a Streptomyces griseus protease. Error bars represent ±S.D.
Multiple regression analysis, with inclusion of more than one incubation period, did not improve estimation of %REP (P < 0.05). Multiple analysis to estimate %REP by harvest date and chemical analysis showed that %REP could only be significantly (P < 0.05) estimated by ash content and harvest date, as expressed by the number of days after 1 April, with: %REP = 52.1 (±9.24) − 0.36 (±0.10) ∗ ash content + 0.15 (±0.03) ∗ days (R2 = 0.60, RSD = 4.1). Remarkably CP did not significantly (P < 0.05) contribute to the estimation of %REP for these grass and grass silage samples. Current results, and those reported by Cone et al. (2002), show that the relationship between in vitro and nylon bag degradability of CP differs between concentrate ingredients and forages, with %REP in concentrate ingredients being best estimated by %U1 and in forages by %U24. This is consistent with De Boever et al. (1997), who suggested that degradation of CP in forages requires more rumen fluid proteolytic activity than that of concentrates.
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5. Conclusions For our grasses and grass silages, the in vitro method, using protease from S. griseus, best estimated nylon bag percentage rumen escape protein with a 24 h incubation period (R2 = 0.71, RSD = 3.4) versus 0, 1, or 6 h. References Aufrère, J., Cartailler, D., 1988. Mise au point d’une méthode de laboratoire de prévision de la dégradabilité des protéines alimentaires des aliments concentrés dans le rumen. Ann. Zootechn. 37, 255–270. Aufrère, J., Graviou, D., Demarquilly, C., Vérité, R., Michalet-Doreau, B., Chapoutot, P., 1991. Predicting in situ degradability of feeds proteins in the rumen by two laboratory methods (solubility and enzymatic degradation). Anim. Feed Sci. Technol. 33, 97–116. Cone, J.W., Van Gelder, A.H., Steg, A., Van Vuuren, A.H., 1996. Prediction of in situ rumen escape protein from in vitro incubations with protease from Streptomyces griseus. J. Sci. Food Agric. 72, 120–126. Cone, J.W., Kamman, A.A., Van Gelder, A.H., Hindle, V.A., 2002. Rumen escape protein in concentrate ingredients determined with the nylon bag and enzymatic techniques. Anim. Feed Sci. Technol. 97, 247–254. De Boever, J.L., Cottyn, B.G., Vanacker, J.M., Boucque, C.V., 1997. Potential of solubility, enzymatic methods and NIRS to predict in situ rumen escape protein. Neth. J. Agric. Sci. 45, 291–306. Krishnamoorthy, U., Sniffen, C.J., Stern, M.D., Van Soest, P.J., 1983. Evaluation of a mathematical model of rumen digestion and in vitro simulation of rumen proteolysis to estimate rumen undegraded nitrogen content of feedstuffs. Br. J. Nutr. 50, 555–568. Licitra, G., Lauria, F., Carpino, S., Schadt, I., Sniffen, C.J., Van Soest, P.J., 1998. Improvement of the Streptomyces griseus method for degradable protein in ruminant feeds. Anim. Feed Sci. Technol. 72, 1–10. Licitra, G., Van Soest, P.J., Schadt, I., Carpino, S., Sniffen, C.J., 1999. Infleunce of the concentration of the protease from Streptomyces griseus relative to ruminal protein degradability. Anim. Feed Sci. Technol. 77, 99–113. Mathis, C.P., Cochran, R.C., Vanzant, E.S., Abdelgadir, I.E.O., Heldt, J.S., Olson, K.C., Johnson, D.E., Caton, J., Faulkner, D., Horn, G., Paisley, S., Mass, R., Moore, K., Halgerson, J., 2001. A collaborative study comparing an in situ protocol with single time-point enzyme assays for estimating ruminal protein degradability of different forages. Anim. Feed Sci. Technol. 93, 31–42. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci., Camb. 92, 499–503. Robinson, P.H., Fadel, J.G., Tamminga, S., 1986. Evaluation of mathematical models to describe neutral detergent residue in terms of its susceptibility to degradation in the rumen. Anim. Feed Sci. Technol. 15, 249–271. Tamminga, S., Van Straalen, W.M., Subnel, A.P.J., Meijer, R.G.M., Steg, A., Wever, C.J.G., Blok, M.C., 1994. The Dutch protein evaluation system: the DVE/OEB-system. Livest. Prod. Sci. 40, 139–155. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Van Vuuren, A.M., Bergsma, F., Krol-Kramer, F., Van Beers, J.A.C., 1989. Effects of addition of cell wall degrading enzymes on the chemical composition and in sacco degradation of grass silage. Grass Forage Sci. 44, 223–230. Van Vuuren, A.M., Van der Koelen, C.J., Valk, H., De Visser, H., 1993. Effects of partial replacement of ryegrass by low protein feeds on rumen fermentation and nitrogen loss by dairy cows. J. Dairy Sci. 76, 2982–2993. Vérité, R., Michalet-Doreau, B., Chapoutot, P., Peyraud, J.L., Poncet, C., 1987. Révision du système des protéines digestibles dans l’intestin (PDI). Bull. Tech. CRZV Theix INRA 70, 19–34.
Rumen escape protein in grass and grass silage determined with a nylon bag and an enzymatic technique J.W. Cone∗ , A.H. van Gelder, A.A. Mathijssen-Kamman, V.A. Hindle Nutrition and Food, Animal Sciences Group of Wageningen UR, P.O. Box 65, NL-8200 AB Lelystad, The Netherlands Received 27 February 2003; received in revised form 18 June 2003; accepted 5 August 2003
Abstract Rumen escape protein (REP) was determined for six grasses and 16 grass silages using a nylon bag technique and an in vitro technique using a proteolytic enzyme preparation of Streptomyces griseus. In vitro, the samples were incubated for 0, 1, 6 and 24 h. The highest correlation observed between percentage REP (%REP), determined with the nylon bag technique, and the in vitro undegradable fraction (U) occurred at 24 h of incubation (%U24; R2 = 0.71). Shorter incubation periods weakened the relationship (R2 = 0.46–0.59). Percentage REP could be estimated from %U24 as %REP = 3.3 (±4.02) + 1.31 (±0.19) ∗ %U24. For the incubation periods investigated, results show that the highest relationship with %REP occurred with an incubation period of 24 h for these grass and grass silage samples. © 2003 Elsevier B.V. All rights reserved. Keywords: Nylon bag technique; Protease; Rumen escape protein; Streptomyces griseus
1. Introduction Many protein evaluation systems for ruminants, such as the Dutch DVE/OEB system (Tamminga et al., 1994) require data obtained from a nylon bag technique. Nylon bag techniques divide feed protein into a washout fraction (W), a rumen undegradable fraction Abbreviations: CP, crude protein (6.25 ∗ N); D, potentially rumen degradable fraction; DM, dry matter; kd , rumen degradation rate in % h−1 ; N, nitrogen; NDF, neutral detergent fibre; REP, rumen escape protein; U, undegradable fraction; %U, percentage in vitro undegradable N; W, washout fraction ∗ Corresponding author. Tel.: +31-320-237354; fax: +31-320-237320. E-mail address: [email protected] (J.W. Cone). 0377-8401/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2003.08.004
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(U) and a potentially rumen degradable insoluble fraction (D = 100 − [W + U]). The degradation rate (kd ) of D can be calculated from the rate of disappearance of nitrogen (N) from the nylon bags (Robinson et al., 1986). Assuming a ruminal passage rate, the percentage rumen escape protein (%REP) can be calculated by the equation of Ørskov and McDonald (1979). An enzymatic technique, using protease from Streptomyces griseus was described by Krishnamoorthy et al. (1983) as a laboratory technique to determine %REP, which is suitable for rapid screening of samples. The technique was further developed by Aufrère et al. (1991) determining N hydrolysis after 1 and 24 h incubation at pH 8.0, and using correction factors for groups of feedstuffs. Cone et al. (1996) simplified this method using the undegradable fraction after 24 h of incubation without correction factors. Cone et al. (1996) used samples of concentrates, concentrate ingredients and forages. However, in a later study, Cone et al. (2002) showed that for concentrate ingredients, the highest correlation was observed between %REP determined with a nylon bag technique and the in vitro undegradable fraction after 1 h of incubation. This raised the question of which in vitro incubation period has the highest relationship with %REP, based on a nylon bag technique for grass and grass silage samples. 2. Materials and methods 2.1. Feed samples and chemical analysis Grass samples were harvested after growth of 3 weeks on different dates in 1996 and 1997 (Table 1). Grass, predominantly Lolium perenne, was grown on a clay soil (Lelystad, The Netherlands) and on a sandy soil (Cranendonck, The Netherlands). Pastures were fertilised with 150 or 300 kg N/ha per year. Harvested samples were stored at −20 ◦ C for later nylon bag incubations or freeze dried and ground to pass a 1 mm screen for chemical analysis and in vitro incubations with protease of S. griseus. At harvest, sub-samples of grass were wilted at 25 or 45% dry matter (DM) and ensiled in 30 l plastic containers. After 8 weeks at 17 (±1) ◦ C, containers were stored at 3 ◦ C until use. Sub-samples from the silages were used for nylon bag incubations, while other sub-samples were freeze dried and ground (1 mm) for chemical and in vitro analysis. Dry matter content was determined after 4 h at 103 ◦ C (ISO 6496) and ash after 3 h at 550 ◦ C (ISO 5984). Crude protein (CP) was determined using a Kjeldahl method (ISO 5983). Sugars were measured colorimetrically, as described by Van Vuuren et al. (1993), after extraction with 40% ethanol followed by treatment with 0.023 M HCl to hydrolyse the bonds between sugar molecules. Neutral detergent fibre (NDF) was determined by a modified method of Van Soest et al. (1991), without use of sodium sulphite. After boiling with ND, the residue was incubated in 60 ml Na–K–phosphate buffer (pH 7.0) for 15 min at 40 ◦ C with 15 mg amylase (Thermamyl 120, NOVO Nordisk, Bagsvaerd, Denmark) and 0.25 ml protease (Alcalase 2.4 l, NOVO Nordisk, Bagsvaerd, Denmark) to remove residues of starch and protein. Lipids were then removed by washing with acetone until the acetone was colourless. NDF is expressed without residual ash.
J.W. Cone et al. / Animal Feed Science and Technology 111 (2004) 1–9
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Table 1 Chemical compositiona (g/kg DM) of the grasses and grass silagesb Soil type
Year
N-fertilization (kg/ha per year)
Harvest date
DM (g/kg)
Ash (g/kg DM)
CP (g/kg DM)
NDF (g/kg DM)
Sugars (g/kg DM)
Grass Clay
1996
150 150 300 300
17 June 18 July 3 June 9 September
227 206 187 168
79 99 85 100
138 169 241 269
513 401 417 436
172 204 137 62
Sand
1997
300 300
17 June 30 June
163 169
103 88
292 156
399 431
78 226
187
92
211
433
146
1997
Mean grass Silage ± 25% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
237 275 227 331 237
93 110 96 106 111
152 190 245 157 281
500 401 412 423 381
30 63 18 109 17
150 300 300
4 August 17 June 30 June
294 261 288
90 106 112
128 300 213
524 358 484
108 51 18
269
103
208
435
52
Mean silage 25% Silage ± 45% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
446 509 431 487 455
89 111 95 105 108
152 192 243 156 275
514 419 429 425 401
93 115 48 133 32
150 300 300
4 August 17 June 30 June
489 502 571
87 109 102
132 305 213
544 391 498
101 77 66
486
101 1.0
209 1.6
453 3.0
83 3.1
Mean silage 45% S.E.M. a b
All samples analysed in duplicate. Pastures were fertilised with 150 or 300 kg N/ha per year.
2.2. In situ nylon bag measurements Grass and silage samples were thawed and cut with a paper guillotine to lengths of approximately 3 cm. CP degradation was determined with the nylon bag technique of Ørskov and McDonald (1979) as modified by Van Vuuren et al. (1989). The W fraction was defined as the fraction disappearing by washing with cold tap water in a washing machine for 45 min.
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Disappearance of N from feed samples (5 g DM) in nylon bags was determined after 3, 6, 12, 24, 48 and 336 h of rumen incubation in triplicate in each of three cows (Van Vuuren et al., 1989). The cows received daily two meals of 8 kg DM, composed of 35% grass silage, 35% maize silage and 30% concentrate. The U fraction was determined as the residual fraction after 336 h of incubation in the rumen. W and U were used as fixed values and the residues were fitted to a first order degradation model (Robinson et al., 1986). Calculations were without a degradation lag time. The %REP was calculated from the nylon bag incubations, assuming a rumen passage rate of 4.5% h−1 (Vérité et al., 1987) and the degradable (D) fraction to be 100 − [W + U]. In the silage samples, 5% of the W fraction was regarded as rumen escape protein according to the Dutch DVE/OEB system (Tamminga et al., 1994). 2.3. In vitro measurements using protease Soluble N was measured after 0, 1, 6 and 24 h of incubation with enzymes in borate/phosphate buffer at pH 8.0 as described by Aufrère and Cartailler (1988) and Cone et al. (1996). The protease from S. griseus (type XIV, Sigma P-5147, St. Louis, MO, USA) was used in a concentration of 20 mg in 1 l buffer. Tetracyclin (1 mg/l, Sigma N-3503) and nystatin (10 mg/l, Sigma T-3258) were added to the buffer to prevent microbial growth. Based on the N content, and soluble N, the percentage of undegradable N (%U) was calculated for the indicated incubation periods (%U0, %U1, %U6 and %U24). 3. Results 3.1. Chemical analysis Ash content ranged from 79 to 112 g/kg DM, CP from 132 to 305 g/kg DM, NDF from 358 to 544 g/kg DM and sugars from 17 to 226 g/kg DM (Table 1). There was no marked influence of ensiling on the chemical composition, with the exception of decreased sugar content in the silages. 3.2. Nylon bag determination of %REP The W fraction of protein ranged from 16% for a grass sample (sandy soil, 1997, 300 kg N/ha per year) to 66% for the silage counterpart (25% DM) of the same sample. U ranged from 7 to 8% for the fresh and silage samples harvested 17 June 1997 on the sandy soil (300 kg N) to 29 to 30% for the silage samples harvested on 4 August 1997 on the sandy soil (150 kg N; Table 2). Data from Table 2 were used to calculate %REP, which ranged from 16.8% to nearly 42%, and was on average lowest in the 25% DM silage samples (27.0%) and highest in the fresh samples (34.9%). 3.3. Enzymatic protein degradation The %U0 (insolubility of CP, Table 3) in the buffer with pH 8.0, without incubation with protease, was highest in fresh grass samples with an average of 58.2% and maximum 66.9%.
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Table 2 The washout (W) and undegradable (U) fractions and degradation rates (kd ) of CP determined with a nylon bag techniquea Soil type
Year
N-fertilizationb (kg/ha per year)
Harvest date
W (% of OM)
U (% of OM)
kd (% h−1 )
%REPc (% of CP)
1996
150 150 300 300
17 June 18 July 3 June 9 September
31 17 28 32
26 12 11 21
14.6 11.2 9.7 5.6
36.1 32.4 30.3 41.9
300 300
17 June 30 June
16 32
8 15
8.4 8.1
34.5 33.9
26.9
15.5
9.6
34.9
Grass Clay
1997 Sand
1997
Mean grass Silage ± 25% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
63 60 62 40 52
21 14 11 22 22
20.3 7.4 14.6 9.8 39.7
27.1 26.8 20.5 36.0 27.2
150 300 300
4 August 17 June 30 June
32 66 53
29 7 19
39.4 14.1 18.6
34.6 16.8 27.1
53.5
18.1
20.5
27.0
Mean silage 25% Silage ± 45% DM Clay
1996
1997 Sand
Mean silage 45% S.E.M.
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
53 55 50 23 38
20 17 12 22 21
11.2 8.0 12.5 15.0 10.1
30.4 29.8 24.6 35.8 35.5
150 300 300
4 August 17 June 30 June
27 41 39
30 8 17
14.0 8.9 17.9
41.8 27.2 27.8
40.8 2.2
18.4 2.3
12.2 2.60
31.6 –
a
All samples analysed in triplicate. Pastures were fertilised with 150 or 300 kg N/ha per year. c %REP was calculated from mean nylon bag parameters, using a passage rate of 4.5% h−1 . For silages 5% of W was assumed to be REP. b
The average %U0 was lowest in the 25% DM silage samples (30.8%), with a minimum of 23.0% (Table 3). The average %U0 of the 45% DM silage samples in the buffer with pH 8.0 was 37.6%. After 1 h in vitro, a considerable part (35.7%) of the insoluble CP in the fresh grass samples was degraded. Remaining insoluble CP after 1 h in vitro was, on average, 37.4%
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Table 3 The percentage undegraded protein (%U) after 0, 1, 6 and 24 h of in vitro incubation with a protease of Streptomyces griseusa Soil type
Year
N-fertilizationb (kg/ha per year)
Harvest date
%U0 (% of CP)
%U1 (% of CP)
%U6 (% of CP)
%U24 (% of CP)
1996
150 150 300 300
17 June 18 July 3 June 9 September
60.1 58.6 56.4 66.9
39.1 34.9 36.5 46.8
30.4 24.9 25.7 38.3
25.4 18.9 20.3 28.3
300 300
17 June 30 June
53.4 53.2
32.5 34.0
23.3 24.4
20.9 23.7
58.2
37.4
27.8
23.1
Grass Clay
1997 Sand
1997
Mean grass Silage ± 25% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
29.6 28.0 27.8 46.8 33.8
25.0 24.2 23.3 43.7 31.3
18.4 18.7 17.6 38.6 27.0
16.4 21.1 16.3 20.9 20.6
150 300 300
4 August 17 June 30 June
32.8 24.7 23.0
30.5 23.3 22.5
26.6 21.0 18.8
24.2 14.0 14.1
30.8
27.9
23.4
18.3
Mean silage 25% Silage ± 45% DM Clay
1996
1997 Sand
1997
150 150 300 300 300
17 June 18 July 3 June 5 August 9 September
32.2 33.3 30.0 47.4 40.7
28.9 29.7 25.1 42.9 38.9
19.7 21.9 18.1 39.1 32.4
19.1 24.0 15.6 25.6 24.0
150 300 300
4 August 17 June 30 June
37.9 41.0 36.6
35.6 37.7 35.2
29.5 28.5 29.6
26.5 21.3 21.1
37.6 1.04
34.2 1.51
27.4 1.58
22.1 1.48
Mean silage 45% S.E.M. a b
All samples analysed in duplicate. Pastures were fertilised with 150 or 300 kg N/ha per year.
of total CP in the grass samples, ranging from 32.5 to 46.8%. After 1 h, CP degradation continued resulting in an average insoluble CP content of 23.1% after 24 h in vitro. After 24 h in vitro, on average, 60.3% of the insoluble CP (100 − %U0) was degraded in the grasses. The ensiled grass samples contained much less insoluble CP than fresh (non-ensiled) grasses. The %U0 was, on average, 30.8% for the 25% DM silages and 37.6% for the 45% DM silages. As a consequence, degradation of CP with Streptomyces proteases occurred more gradually in the silages than in the fresh grass samples. However, the %U24 was, on
J.W. Cone et al. / Animal Feed Science and Technology 111 (2004) 1–9
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Table 4 Relationships between the nylon bag determined rumen escape protein (%REP) and percentage (%U) undegraded protein after 0, 1, 6 and 24 h of in vitro incubation with a Streptomyces griseus protease Incubation period (h) 0 1 6 24
Regression equation ∗ %U0
17.4 (± 3.40) + 0.33 (± 0.08) 8.9 (± 4.19) + 0.67 (± 0.12) ∗ %U1 14.2 (± 3.96) + 0.64 (± 0.15) ∗ %U6 3.3 (± 4.02) + 1.31 (± 0.19) ∗ %U24
R2
RSD
0.46 0.59 0.48 0.71
4.7 4.1 4.6 3.4
average, only slightly lower in the silages (18.3–22.1%) than in the fresh grass samples (23.1%). 3.4. Correlations between nylon bag and in vitro protein degradation Relationships between %REP determined in nylon bags (Table 2), and the in vitro determined %U after 0, 1, 6 and 24 h of incubation (Table 3), are in Table 4. %REP showed the highest correlation with %U after 24 h and the lowest after 0 h of incubation in vitro (i.e. solubility). Only the regression equation between %REP and %U24 had an intercept that did not differ from zero (Table 4 and Fig. 1). The slopes for all regression equations differed from one. Fig. 1 shows the relationships between the calculated %REP, determined with the nylon bag technique, and %U, obtained after 1 or 24 h incubation in vitro. From the figure, it is evident that the grass samples did not behave different from the silage samples. 4. Discussion Aufrère et al. (1991) estimated nylon bag determined %REP from in vitro enzymatic degradation after 1 and 24 h of incubation. Cone et al. (1996) showed that nylon bag estimated %REP was best correlated with %U24, using a mixed data set of mixed concentrates, concentrate ingredients and forages. Recently, Cone et al. (2002) showed that, for 26 concentrate ingredients, %REP was best estimated after only 1 h of incubation with S. griseus protease, suggesting that %REP in concentrate ingredients is mainly determined by its CP solubility. Licitra et al. (1998) showed for a number of feed samples a decreased residual-N by extending the incubation period from 8 to 18 h. Extending the incubation period from 18 to 24 or 30 h did not increase the solubility of N for the majority of the feed samples (Licitra et al., 1998). Licitra et al. (1999) showed that best regressions with in situ N degradation was obtained using a short incubation period (4 h) with high enzyme concentrations or prolonged incubation periods (36–48 h) with low enzyme concentrations. These results were confirmed by Mathis et al. (2001), investigating ruminal protein degradability of five forages. Our results show that for grass and grass silage samples incubated in nylon bags, %REP was best estimated by %U after 24 h of incubation in vitro, as the relationship between %REP and %U24 had the highest R2 , lowest RSD and an intercept that did not differ from zero. This suggests that for the investigated forage samples, %REP is determined partly by solubility and partly by degradation of CP by the S. griseus protease.
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50
Nylon bag technique estimated rumen escape protein (% of CP)
40
30
20
10 10
20
30
40
50
In vitro undegraded CP (% of CP) Fig. 1. Relationship between percentage rumen escape protein determined with a nylon bag technique and the in vitro percentage undegraded CP of grasses (, 䉱) and grass silages (䊊, 䊉) after 1 (䊊, ) or 24 (䊉, 䉱) h of incubation with a Streptomyces griseus protease. Error bars represent ±S.D.
Multiple regression analysis, with inclusion of more than one incubation period, did not improve estimation of %REP (P < 0.05). Multiple analysis to estimate %REP by harvest date and chemical analysis showed that %REP could only be significantly (P < 0.05) estimated by ash content and harvest date, as expressed by the number of days after 1 April, with: %REP = 52.1 (±9.24) − 0.36 (±0.10) ∗ ash content + 0.15 (±0.03) ∗ days (R2 = 0.60, RSD = 4.1). Remarkably CP did not significantly (P < 0.05) contribute to the estimation of %REP for these grass and grass silage samples. Current results, and those reported by Cone et al. (2002), show that the relationship between in vitro and nylon bag degradability of CP differs between concentrate ingredients and forages, with %REP in concentrate ingredients being best estimated by %U1 and in forages by %U24. This is consistent with De Boever et al. (1997), who suggested that degradation of CP in forages requires more rumen fluid proteolytic activity than that of concentrates.
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5. Conclusions For our grasses and grass silages, the in vitro method, using protease from S. griseus, best estimated nylon bag percentage rumen escape protein with a 24 h incubation period (R2 = 0.71, RSD = 3.4) versus 0, 1, or 6 h. References Aufrère, J., Cartailler, D., 1988. Mise au point d’une méthode de laboratoire de prévision de la dégradabilité des protéines alimentaires des aliments concentrés dans le rumen. Ann. Zootechn. 37, 255–270. Aufrère, J., Graviou, D., Demarquilly, C., Vérité, R., Michalet-Doreau, B., Chapoutot, P., 1991. Predicting in situ degradability of feeds proteins in the rumen by two laboratory methods (solubility and enzymatic degradation). Anim. Feed Sci. Technol. 33, 97–116. Cone, J.W., Van Gelder, A.H., Steg, A., Van Vuuren, A.H., 1996. Prediction of in situ rumen escape protein from in vitro incubations with protease from Streptomyces griseus. J. Sci. Food Agric. 72, 120–126. Cone, J.W., Kamman, A.A., Van Gelder, A.H., Hindle, V.A., 2002. Rumen escape protein in concentrate ingredients determined with the nylon bag and enzymatic techniques. Anim. Feed Sci. Technol. 97, 247–254. De Boever, J.L., Cottyn, B.G., Vanacker, J.M., Boucque, C.V., 1997. Potential of solubility, enzymatic methods and NIRS to predict in situ rumen escape protein. Neth. J. Agric. Sci. 45, 291–306. Krishnamoorthy, U., Sniffen, C.J., Stern, M.D., Van Soest, P.J., 1983. Evaluation of a mathematical model of rumen digestion and in vitro simulation of rumen proteolysis to estimate rumen undegraded nitrogen content of feedstuffs. Br. J. Nutr. 50, 555–568. Licitra, G., Lauria, F., Carpino, S., Schadt, I., Sniffen, C.J., Van Soest, P.J., 1998. Improvement of the Streptomyces griseus method for degradable protein in ruminant feeds. Anim. Feed Sci. Technol. 72, 1–10. Licitra, G., Van Soest, P.J., Schadt, I., Carpino, S., Sniffen, C.J., 1999. Infleunce of the concentration of the protease from Streptomyces griseus relative to ruminal protein degradability. Anim. Feed Sci. Technol. 77, 99–113. Mathis, C.P., Cochran, R.C., Vanzant, E.S., Abdelgadir, I.E.O., Heldt, J.S., Olson, K.C., Johnson, D.E., Caton, J., Faulkner, D., Horn, G., Paisley, S., Mass, R., Moore, K., Halgerson, J., 2001. A collaborative study comparing an in situ protocol with single time-point enzyme assays for estimating ruminal protein degradability of different forages. Anim. Feed Sci. Technol. 93, 31–42. Ørskov, E.R., McDonald, I., 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci., Camb. 92, 499–503. Robinson, P.H., Fadel, J.G., Tamminga, S., 1986. Evaluation of mathematical models to describe neutral detergent residue in terms of its susceptibility to degradation in the rumen. Anim. Feed Sci. Technol. 15, 249–271. Tamminga, S., Van Straalen, W.M., Subnel, A.P.J., Meijer, R.G.M., Steg, A., Wever, C.J.G., Blok, M.C., 1994. The Dutch protein evaluation system: the DVE/OEB-system. Livest. Prod. Sci. 40, 139–155. Van Soest, P.J., Robertson, J.B., Lewis, B.A., 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583–3597. Van Vuuren, A.M., Bergsma, F., Krol-Kramer, F., Van Beers, J.A.C., 1989. Effects of addition of cell wall degrading enzymes on the chemical composition and in sacco degradation of grass silage. Grass Forage Sci. 44, 223–230. Van Vuuren, A.M., Van der Koelen, C.J., Valk, H., De Visser, H., 1993. Effects of partial replacement of ryegrass by low protein feeds on rumen fermentation and nitrogen loss by dairy cows. J. Dairy Sci. 76, 2982–2993. Vérité, R., Michalet-Doreau, B., Chapoutot, P., Peyraud, J.L., Poncet, C., 1987. Révision du système des protéines digestibles dans l’intestin (PDI). Bull. Tech. CRZV Theix INRA 70, 19–34.