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Estimation of digestibility, metabolizable energy and net energy of forage by a cellulase method
Livestock Production Science, 5 (1978) 373--377
373
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
ESTIMATION OF DIGESTIBILITY, METABOLIZABLE ENERGY AND NET ENERGY OF F O R A G E BY A CELLULASE METHOD
M. K I R C H G E S S N E R
and R.J. K E L L N E R
lnstitut f~r Errdlhrungsphysiologie der Technischen Universit~it Milnchen in FreisingWeihenstephan (Federal Republic of Germany)
(Received 1 December 1977)
ABSTRACT Kirchgessner, M. and Kellner, R.J., 1978. Estimation of digestibility, metabolizable energy and net energy of forage by a cellulase method. Livest. Prod. Sci., 5: 373--377. As an alternative to existing methods for digestibility determination using rumen juice, a cellulase method is proposed for routine use, which is easy, cheap and sufficiently precise. From material comprising 47 green and preserved forages of known digestibility in vivo regression equations have been derived for the estimation of the organic matter digestibility, the metabolizable energy, the net energy-fat (NEFf) and the starch equivalents.
INTRODUCTION
Among the in-vitro m e t h o d s for determination of the digestibility by the ruminant animal, the two-stage m e t h o d of Tilley and Terry (1963) has found the widest application. This procedure has some disadvantages, such as the housing o f fistulated animals and the varying activity of rumen juice (Kellner and Kirchgessner, 1976a). Such difficulties m a y be avoided if the rumen inoculum is replaced by enzyme preparations with cellulolytic activity. Several approaches (for references see Kellner and Kirchgessner, 1977) resulted in appreciably lower digestibilities than those obtained with the animal. Therefore, a procedure has been developed, to ascertain the level of in-vivo digestibility (Kellner and Kirchgessner, 1976b). The essential of this procedure is the introduction of a swelling step using dilute hydrochloric acid. Swelling is an i m p o r t a n t factor in the improvement o f cellulolysis, because it causes the pores of plant material to enlarge, allowing the enzyme to penetrate the cellulose miceUes. METHOD The m e t h o d involves the swelling of the ground feed sample with boiling
374
2-N HC1, and thereafter incubation with both cellulase and pepsin. A full description is given in the original publications by Kellner and Kirchgessner, (1976b, 1977). To evaluate this cellulase method, the digestibilities obtained must be compared with those obtained in vivo. For this purpose, 4"/samples of fresh and dried forages of known digestibility (from digestion trials with wethers) were available. The in-vitro digestibility of the same 47 forages was also determined by the two-stage method of Tilley and Terry (1963). Thus, these results could be compared with those of the cellulase method. The final aim of these determinations of digestibility was the energy evaluation of feeds. Therefore, the digestible nutrients determined in vivo were used to compute the metabolizable energy (ME), according to the Rostock equation (Schiemann et al., 1971), and the net energy, expressed both as starch equivalents (SE) (g/kg DM) and as net energy-fat (NEFr) (Schiemann et al., 1971). These energy values served as dependent variables (Y) in the derivation of regression equations by step-wise multiple regression analysis. Besides the 'ceUulase
375
TABLE I Regression equations for estimation o f organic matter digestibility (DOM) DOM 16.7 -3.7 101.5 102.2
+ + -
0.80 1.04 1.13 0.98
IV-DOM 3 DOM-cellulase crude fibre ADF 4
RSD 1
% RSD 2
r2
2.1 2.5 3.4 3.2
2.9 3.5 4.7 4.5
0.92 0.88 0.78 0.80
i Residual standard deviation. 2 Residual standard deviation as % mean of the dependent variable. 3 Digestibility o f organic matter by the two-stage method. 4 Acid detergent fibre.
ESTIMATION OF THE METABOLIZABLE ENERGY AND NET ENERGY The struggle for the best choice of systems for the energetic evaluation of f e e d is n o t o f g r e a t i m p o r t a n c e in r e s p e c t t o t h e c e l l u l a s e m e t h o d a n d t h e o t h e r m e t h o d s d i s c u s s e d h e r e . I t is p o s s i b l e t o e s t i m a t e t h e v a l u e s f o r m e t a b o l i z a b l e e n e r g y a n d n e t e n e r g y w i t h all t h e s e m e t h o d s , b u t w i t h d i f f e r e n t accuracy. From the available data: 'two-stage-digestibility', 'cellulase-digestibility', the crude nutrient contents, and the cell-wall fractions, many regression equations can be derived with stepwise regression analysis. A measure for a c r i t i c a l e x a m i n a t i o n o f s u c h e q u a t i o n s is t h e r e l a t i o n s h i p b e t w e e n t h e d i g e s t i b i l i t y in v i v o a n d t h e c o r r e s p o n d i n g e n e r g y v a l u e . T a b l e I I gives s o m e s e l e c t e d r e g r e s s i o n e q u a t i o n s f o r t h e e s t i m a t i o n o f metabolizable energy in terms of MJ/kg DM. As expected, the best estimaTABLE
II
Regression equations for estimation of metabolizable energy (ME) ME (MJ/kg DM)
RSD
% RSD
r2
- 0 . 4 8 + 0.145 2.03 ÷ 0.115 2.33 + 0.098 fat 14.35 - 0.168 12.81 0.141 crude fat - 1 . 1 6 + 0.152 --0.45 + 0.134 crude fat 14.41 - 0.144
0.22 0.39
2.2 3.9
0.96 0.87
0.33 0.50
3.4 5.0
0.90 0.78
0.46 0.41
4.7 4.1
0.82 0.86
0.39 0.50
3.9 5.0
0.87 0.79
-
DOM IV-DOM IV-DOM + 0.207 crude crude fibre crude fibre + 0.206 DOM-cellulase DOM-cellulase + 0.157 ADF
376
tion is given by digestibility of organic matter in vivo. Thus, the values of 2.2% for the percentage RSD, and 0.96 for the coefficient of determination serve as measures for the equations derived from laboratory data. With a small loss of accuracy, the estimation of the metabolizabl~ energy is also possible by the use of fibre values (Kirchgessner and Kellner, 1977). Equations for the estimation of NEFr and SE are also possible. The use of the cellulase m e t h o d for the estimation of the NEFr or the SE results in a percentage RSD of 6.4 and 6.2%, respectively (Kellner and Kirchgessner, 1976c). The estimation with the two-stage digestibility gives a better value in the case of NEFr (5.4% RSD), whereas in the estimation of SE, a percentage RSD of only 8.1% is found (Kellner and Kirchgessner, 1976d). For routine use, especially for advisory purposes, the cellulase method with the possible addition of the crude fat content and the crude protein content is recommended, because it is easy, cheap and sufficiently precise. ACKNOWLEDGEMENT
This study was c o n d u c t e d with the financial support of the German Scientific Foundation, SFB 141"Produktionstechniken in der Rinderhaltung". REFERENCES Goering, H.K. and Van Soest, P.J., 1970. Forage Fiber Analyses (apparatus, reagents, procedures, and some applications). Agriculture Handbook No. 379, U.S. Dep. Agric. Agricultural Research Service, 20 pp. Kellner, R.J. and Kirchgessner, M., 1976a. Zur in vitro Bestimmung der Verdaulichkeit yon Griin- und Rauhfutter. Wirtschaftseigene Futter, 22: 157--166. KeUner, R.J. and Kirchgessner, M., 1976b. Zur Methodik der in vitro Verdaulichkeitsbestimmung yon Griin- und Rauhfutter mit Cellulase. Landwirtsch. Forsch., 29: 204--210. Kellner, R.J. and Kirchgessner, M., 1976c. Zur Sch~tzung der Nettoenergie yon Griinund Rauhfutter mit Hilfe der Rohn~hrstoffe und der nach der Cellulase-Methode bestimmten Verdaulichkeit. Z. Tierphysiol., Tierern~/hr. Futtermittelkd., 37: 208--212. KeUner, R.J. and Kirchgessner, M., 1976d. Zur Sch~tzung der Nettoenergie yon Griinund Rauhfutter mit Hilfe der Rohn~hrstoffe und der in-vitro-Verdaulichkeit. Z. Tierphysiol., Tierern~'hr. Futtermittelkd., 37: 201--207. Kellner, R.J. and Kirchgessner, M., 1977. Estimation of forage digestibility by a cellulase method. Z. Tierphysiol., Tierern//hr. Futtermittelkd., 39: 9--16. Kirchgessner, M. and Kellner, R.J., 1977. Zur Sch~tzung der umsetzbaren Energie yon Griin- und Rauhfutter mit einfachen Kenndaten. Z. Tierphysiol., Tierern~/hr. Futtermittelkd., 38: 297--301. Kirchgessner, M., Kellner, R.J., Roth, F.X. and Ranfft, K., 1977. Zur Sch~'tzung des Futterwertes mittels Rohfaser und der Zellwandfraktionen der Detergentien-Analyse. Landwirtsch. Forsch., 30: 245--250. Schiemann, R., Nehring, K., Hoffmann, L., Jentsch, W. and Chudy, A., 1971. Energetische Futterbewertung und Energienormen. VEB Deutscher Landwirtschaftsverlag, Berlin, 344 pp. Tilley, J.M.A. and Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc., 18: 104--111.
377
RESUME Kirchgessner, M. et Kellner, R.J., 1978. Estimation de la digestibilit~ et des teneurs en ~nergie metabolisable et ~nergie nette des fourrages ~ l'aide d ' u n e metbode ~ la cellulase. Livest. Prod. Sci., 5 : 3 7 3 - - 3 7 7 (en anglais). En remplacement des m4thodes actuelles d'estimation de la digestibilitd~ l'aide de liquide du rumen, on propose pour les ~tudes de s~rie une m~thode ~ la cellulase qui est facile,bon march~ et suffisamment precise. A partir d'un ensemble de 47 fourrages verts ou conservds dont la digestibilit~in vivo ~tait connue, on a calcul~ les dquations de r~gression estimant la digestibilit$de la mati~re organique, la teneur en dnergie m~tabolisable, la teneur en ~nergie nette d'engraissement et la valeur amidon. KURZFASSUNG Kirchgessner, M. und Kellner, R.J., 1978. Sch~tzung der Verdaulichkeit, der umsetzbaren Energie und der Nettoenergie mittels einer Cellulasemethode. Livest. Prod. Sci., 5: 373--377 (in Englisch). Als Alternative zu bestehenden Methoden der fiirVerdaulichkeitsermittlung, bei denen Pansensaft verwendet wird, wird fiirden Einsatz in der Futtermitteluntersuchung eine Cellulaseverdauungsmethode vorgeschlagen mit einfacher Handhabung bei hinreichender Genauigkeit. Aus einem Material yon 47 Griin- und Rauhfutterstoffen mit bekannter Verdaulichkeit in vivo, wurden Regressionsgleichungen gewonnen fiirdie Scb~'tzung der Verdaulichkeit der organischen Substanz, der umsetzbaren Energie, der Nettoenergie-FettRind und der St~/rkeeinheiten.
373
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
ESTIMATION OF DIGESTIBILITY, METABOLIZABLE ENERGY AND NET ENERGY OF F O R A G E BY A CELLULASE METHOD
M. K I R C H G E S S N E R
and R.J. K E L L N E R
lnstitut f~r Errdlhrungsphysiologie der Technischen Universit~it Milnchen in FreisingWeihenstephan (Federal Republic of Germany)
(Received 1 December 1977)
ABSTRACT Kirchgessner, M. and Kellner, R.J., 1978. Estimation of digestibility, metabolizable energy and net energy of forage by a cellulase method. Livest. Prod. Sci., 5: 373--377. As an alternative to existing methods for digestibility determination using rumen juice, a cellulase method is proposed for routine use, which is easy, cheap and sufficiently precise. From material comprising 47 green and preserved forages of known digestibility in vivo regression equations have been derived for the estimation of the organic matter digestibility, the metabolizable energy, the net energy-fat (NEFf) and the starch equivalents.
INTRODUCTION
Among the in-vitro m e t h o d s for determination of the digestibility by the ruminant animal, the two-stage m e t h o d of Tilley and Terry (1963) has found the widest application. This procedure has some disadvantages, such as the housing o f fistulated animals and the varying activity of rumen juice (Kellner and Kirchgessner, 1976a). Such difficulties m a y be avoided if the rumen inoculum is replaced by enzyme preparations with cellulolytic activity. Several approaches (for references see Kellner and Kirchgessner, 1977) resulted in appreciably lower digestibilities than those obtained with the animal. Therefore, a procedure has been developed, to ascertain the level of in-vivo digestibility (Kellner and Kirchgessner, 1976b). The essential of this procedure is the introduction of a swelling step using dilute hydrochloric acid. Swelling is an i m p o r t a n t factor in the improvement o f cellulolysis, because it causes the pores of plant material to enlarge, allowing the enzyme to penetrate the cellulose miceUes. METHOD The m e t h o d involves the swelling of the ground feed sample with boiling
374
2-N HC1, and thereafter incubation with both cellulase and pepsin. A full description is given in the original publications by Kellner and Kirchgessner, (1976b, 1977). To evaluate this cellulase method, the digestibilities obtained must be compared with those obtained in vivo. For this purpose, 4"/samples of fresh and dried forages of known digestibility (from digestion trials with wethers) were available. The in-vitro digestibility of the same 47 forages was also determined by the two-stage method of Tilley and Terry (1963). Thus, these results could be compared with those of the cellulase method. The final aim of these determinations of digestibility was the energy evaluation of feeds. Therefore, the digestible nutrients determined in vivo were used to compute the metabolizable energy (ME), according to the Rostock equation (Schiemann et al., 1971), and the net energy, expressed both as starch equivalents (SE) (g/kg DM) and as net energy-fat (NEFr) (Schiemann et al., 1971). These energy values served as dependent variables (Y) in the derivation of regression equations by step-wise multiple regression analysis. Besides the 'ceUulase
375
TABLE I Regression equations for estimation o f organic matter digestibility (DOM) DOM 16.7 -3.7 101.5 102.2
+ + -
0.80 1.04 1.13 0.98
IV-DOM 3 DOM-cellulase crude fibre ADF 4
RSD 1
% RSD 2
r2
2.1 2.5 3.4 3.2
2.9 3.5 4.7 4.5
0.92 0.88 0.78 0.80
i Residual standard deviation. 2 Residual standard deviation as % mean of the dependent variable. 3 Digestibility o f organic matter by the two-stage method. 4 Acid detergent fibre.
ESTIMATION OF THE METABOLIZABLE ENERGY AND NET ENERGY The struggle for the best choice of systems for the energetic evaluation of f e e d is n o t o f g r e a t i m p o r t a n c e in r e s p e c t t o t h e c e l l u l a s e m e t h o d a n d t h e o t h e r m e t h o d s d i s c u s s e d h e r e . I t is p o s s i b l e t o e s t i m a t e t h e v a l u e s f o r m e t a b o l i z a b l e e n e r g y a n d n e t e n e r g y w i t h all t h e s e m e t h o d s , b u t w i t h d i f f e r e n t accuracy. From the available data: 'two-stage-digestibility', 'cellulase-digestibility', the crude nutrient contents, and the cell-wall fractions, many regression equations can be derived with stepwise regression analysis. A measure for a c r i t i c a l e x a m i n a t i o n o f s u c h e q u a t i o n s is t h e r e l a t i o n s h i p b e t w e e n t h e d i g e s t i b i l i t y in v i v o a n d t h e c o r r e s p o n d i n g e n e r g y v a l u e . T a b l e I I gives s o m e s e l e c t e d r e g r e s s i o n e q u a t i o n s f o r t h e e s t i m a t i o n o f metabolizable energy in terms of MJ/kg DM. As expected, the best estimaTABLE
II
Regression equations for estimation of metabolizable energy (ME) ME (MJ/kg DM)
RSD
% RSD
r2
- 0 . 4 8 + 0.145 2.03 ÷ 0.115 2.33 + 0.098 fat 14.35 - 0.168 12.81 0.141 crude fat - 1 . 1 6 + 0.152 --0.45 + 0.134 crude fat 14.41 - 0.144
0.22 0.39
2.2 3.9
0.96 0.87
0.33 0.50
3.4 5.0
0.90 0.78
0.46 0.41
4.7 4.1
0.82 0.86
0.39 0.50
3.9 5.0
0.87 0.79
-
DOM IV-DOM IV-DOM + 0.207 crude crude fibre crude fibre + 0.206 DOM-cellulase DOM-cellulase + 0.157 ADF
376
tion is given by digestibility of organic matter in vivo. Thus, the values of 2.2% for the percentage RSD, and 0.96 for the coefficient of determination serve as measures for the equations derived from laboratory data. With a small loss of accuracy, the estimation of the metabolizabl~ energy is also possible by the use of fibre values (Kirchgessner and Kellner, 1977). Equations for the estimation of NEFr and SE are also possible. The use of the cellulase m e t h o d for the estimation of the NEFr or the SE results in a percentage RSD of 6.4 and 6.2%, respectively (Kellner and Kirchgessner, 1976c). The estimation with the two-stage digestibility gives a better value in the case of NEFr (5.4% RSD), whereas in the estimation of SE, a percentage RSD of only 8.1% is found (Kellner and Kirchgessner, 1976d). For routine use, especially for advisory purposes, the cellulase method with the possible addition of the crude fat content and the crude protein content is recommended, because it is easy, cheap and sufficiently precise. ACKNOWLEDGEMENT
This study was c o n d u c t e d with the financial support of the German Scientific Foundation, SFB 141"Produktionstechniken in der Rinderhaltung". REFERENCES Goering, H.K. and Van Soest, P.J., 1970. Forage Fiber Analyses (apparatus, reagents, procedures, and some applications). Agriculture Handbook No. 379, U.S. Dep. Agric. Agricultural Research Service, 20 pp. Kellner, R.J. and Kirchgessner, M., 1976a. Zur in vitro Bestimmung der Verdaulichkeit yon Griin- und Rauhfutter. Wirtschaftseigene Futter, 22: 157--166. KeUner, R.J. and Kirchgessner, M., 1976b. Zur Methodik der in vitro Verdaulichkeitsbestimmung yon Griin- und Rauhfutter mit Cellulase. Landwirtsch. Forsch., 29: 204--210. Kellner, R.J. and Kirchgessner, M., 1976c. Zur Sch~tzung der Nettoenergie yon Griinund Rauhfutter mit Hilfe der Rohn~hrstoffe und der nach der Cellulase-Methode bestimmten Verdaulichkeit. Z. Tierphysiol., Tierern~/hr. Futtermittelkd., 37: 208--212. KeUner, R.J. and Kirchgessner, M., 1976d. Zur Sch~tzung der Nettoenergie yon Griinund Rauhfutter mit Hilfe der Rohn~hrstoffe und der in-vitro-Verdaulichkeit. Z. Tierphysiol., Tierern~'hr. Futtermittelkd., 37: 201--207. Kellner, R.J. and Kirchgessner, M., 1977. Estimation of forage digestibility by a cellulase method. Z. Tierphysiol., Tierern//hr. Futtermittelkd., 39: 9--16. Kirchgessner, M. and Kellner, R.J., 1977. Zur Sch~tzung der umsetzbaren Energie yon Griin- und Rauhfutter mit einfachen Kenndaten. Z. Tierphysiol., Tierern~/hr. Futtermittelkd., 38: 297--301. Kirchgessner, M., Kellner, R.J., Roth, F.X. and Ranfft, K., 1977. Zur Sch~'tzung des Futterwertes mittels Rohfaser und der Zellwandfraktionen der Detergentien-Analyse. Landwirtsch. Forsch., 30: 245--250. Schiemann, R., Nehring, K., Hoffmann, L., Jentsch, W. and Chudy, A., 1971. Energetische Futterbewertung und Energienormen. VEB Deutscher Landwirtschaftsverlag, Berlin, 344 pp. Tilley, J.M.A. and Terry, R.A., 1963. A two-stage technique for the in vitro digestion of forage crops. J. Br. Grassl. Soc., 18: 104--111.
377
RESUME Kirchgessner, M. et Kellner, R.J., 1978. Estimation de la digestibilit~ et des teneurs en ~nergie metabolisable et ~nergie nette des fourrages ~ l'aide d ' u n e metbode ~ la cellulase. Livest. Prod. Sci., 5 : 3 7 3 - - 3 7 7 (en anglais). En remplacement des m4thodes actuelles d'estimation de la digestibilitd~ l'aide de liquide du rumen, on propose pour les ~tudes de s~rie une m~thode ~ la cellulase qui est facile,bon march~ et suffisamment precise. A partir d'un ensemble de 47 fourrages verts ou conservds dont la digestibilit~in vivo ~tait connue, on a calcul~ les dquations de r~gression estimant la digestibilit$de la mati~re organique, la teneur en dnergie m~tabolisable, la teneur en ~nergie nette d'engraissement et la valeur amidon. KURZFASSUNG Kirchgessner, M. und Kellner, R.J., 1978. Sch~tzung der Verdaulichkeit, der umsetzbaren Energie und der Nettoenergie mittels einer Cellulasemethode. Livest. Prod. Sci., 5: 373--377 (in Englisch). Als Alternative zu bestehenden Methoden der fiirVerdaulichkeitsermittlung, bei denen Pansensaft verwendet wird, wird fiirden Einsatz in der Futtermitteluntersuchung eine Cellulaseverdauungsmethode vorgeschlagen mit einfacher Handhabung bei hinreichender Genauigkeit. Aus einem Material yon 47 Griin- und Rauhfutterstoffen mit bekannter Verdaulichkeit in vivo, wurden Regressionsgleichungen gewonnen fiirdie Scb~'tzung der Verdaulichkeit der organischen Substanz, der umsetzbaren Energie, der Nettoenergie-FettRind und der St~/rkeeinheiten.