Skip to main content Accessibility help

The effect of cultivar on the changes in protein quality during wilting and ensiling of red clover (Trifolium pratense L.)

  • M. KRAWUTSCHKE (a1), N. WEIHER (a1), J. THAYSEN (a2), R. LOGES (a1), F. TAUBE (a1) and M. GIERUS (a1)...


Red clover (Trifolium pratense L.) silage usually contains lower contents of non-protein nitrogen (NPN) compared with other forage legumes. This is often attributed to the polyphenol oxidase (PPO) activity in red clover, although in most field studies the PPO activity was not measured. Therefore, a laboratory ensiling experiment with three red clover cultivars and one white clover cultivar as control grown in two management systems (with and without mechanical stress) over 2 consecutive years was conducted. Fresh, wilted and ensiled clover herbage was sampled at four cutting dates per year to determine the crude protein (CP) fractions according to the Cornell Net Carbohydrate and Protein System. The specific PPO activity was measured photometrically in fresh clover leaves. The content of CP fraction A (NPN) increased from fresh over wilted to ensiled clover herbage at the expense of the content of CP fraction B (true protein), irrespective of species, cultivar and year. The most important source of variation for all CP fractions and the calculated rumen-undegradable protein contents was generally the herbage condition, except for CP fraction C (unavailable protein). White clover silage consisted of higher contents of CP fraction A and lower contents of CP fraction B3 in CP compared with red clover silage. As a result, the calculated rumen-undegradable protein content of white clover silage was lower than that of all red clover cultivars. In conclusion, the extent of proteolysis during ensiling among the silages made from the herbage of different red clover cultivars was primarily influenced by the stage of maturity at harvesting and the degree of wilting at ensiling. The variation in specific PPO activity could not be related to the extent of proteolysis.


Corresponding author

*To whom all correspondence should be addressed. Email:


Hide All
Abberton, M. T. & Marshall, A. H. (2005). Progress in breeding perennial clovers for temperate agriculture. Journal of Agricultural Science, Cambridge 143, 117135.
Albrecht, K. A. & Muck, R. E. (1991). Proteolysis in ensiled forage legumes that vary in tannin concentration. Crop Science 31, 464469.
Broderick, G. A., Brito, A. F. & Olmos Colmenero, J. J. (2007). Effects of feeding formate-treated alfalfa silage or red clover silage on the production of lactating dairy cows. Journal of Dairy Science 90, 13781391.
Cavallarin, L., Antoniazzi, S., Tabacco, E. & Borreani, G. (2006). Effect of the stage of growth, wilting and inoculation in field pea (Pisum sativum L.) silages. II. Nitrogen fractions and amino acid compositions of herbage and silage. Journal of the Science of Food and Agriculture 86, 13831390.
Eickler, B., Gierus, M., Kleen, J. & Taube, F. (2011). Specific polyphenol oxidase activity of red clover (Trifolium pratense L.) and its relation with forage quality in field experiments. Acta Agriculturae Scandinavica, Section B – Soil and Plant Science 61, 3949.
Escribano, J., Cabanes, J., Chazarra, S. & García-Carmona, F. (1997). Characterization of monophenolase activity of table beet polyphenol oxidase. Determination of kinetic parameters on the tyramine/dopamine pair. Journal of Agricultural and Food Chemistry 45, 42094214.
Fagerberg, B. (1988). Phenological development in timothy, red clover and lucerne. Acta Agriculturae Scandinavica 38, 159170.
Givens, D. I. & Rulquin, H. (2004). Utilisation by ruminants of nitrogen compounds in silage-based diets. Animal Feed Science and Technology 114, 118.
Grabber, J. H. & Coblentz, W. K. (2009). Polyphenol, conditioning, and conservation effects on protein fractions and degradability in forage legumes. Crop Science 49, 15111522.
Guo, X., Zhou, H., Yu, Z. & Zhang, Y. (2007). Changes in the distribution of nitrogen and plant enzymatic activity during ensilage of lucerne treated with different additives. Grass and Forage Science 62, 3543.
Henderson, A. R., McDonald, P. & Woolford, M. K. (1972). Chemical changes and losses during the ensilage of wilted grass treated with formic acid. Journal of the Science of Food and Agriculture 23, 10791087.
Holm, S. (1979). A simple sequentially rejective multiple test procedure. Scandinavian Journal of Statistics 6, 6570.
Jones, B. A., Satter, L. D. & Muck, R. E. (1992). Influence of bacterial inoculant and substrate addition to lucerne ensiled at different dry matter contents. Grass and Forage Science 47, 1927.
Jones, B. A., Hatfield, R. D. & Muck, R. E. (1995). Screening legume forages for soluble phenols, polyphenol oxidase and extract browning. Journal of the Science of Food and Agriculture 67, 109112.
Kalscheur, K. F., Baldwin, R. L., Glenn, B. P. & Kohn, R. A. (2006). Milk production of dairy cows fed differing concentrations of rumen-degraded protein. Journal of Dairy Science 89, 249259.
Kemble, A. R. & Macpherson, H. T. (1954). Liberation of amino acids in perennial rye grass during wilting. Biochemical Journal 58, 4649.
Kohn, R. A. & Allen, M. S. (1995). Effect of plant maturity and preservation method on in vitro protein degradation of forages. Journal of Dairy Science 78, 15441551.
Kung, L. & Shaver, R. (2001). Interpretation and use of silage fermentation analysis reports. Focus on Forage 3, 15.
Lee, M. R. F., Scott, M. B., Tweed, J. K. S., Minchin, F. R. & Davies, D. R. (2008). Effects of polyphenol oxidase on lipolysis and proteolysis of red clover silage with and without a silage inoculant (Lactobacillus plantarum L54). Animal Feed Science and Technology 144, 125136.
Licitra, G., Hernandez, T. M. & Van Soest, P. J. (1996). Standardization of procedures for nitrogen fractionation of ruminant feeds. Animal Feed Science and Technology 57, 347358.
Matheis, G. & Whitaker, J. R. (1984). Modification of proteins by polyphenol oxidase and peroxidase and their products. Journal of Food Biochemistry 8, 137162.
McKersie, B. D. (1985). Effect of pH on proteolysis in ensiled legume forage. Agronomy Journal 77, 8186.
Merchen, N. R. & Satter, L. D. (1983). Changes in nitrogenous compounds and sites of digestion of alfalfa harvested at different moisture contents. Journal of Dairy Science 66, 789801.
Mustafa, A. F. & Seguin, P. (2003). Ensiling characteristics, ruminal nutrient degradabilities and whole tract nutrient utilization of berseem clover (Trifolium alexandrinum L.) silage. Canadian Journal of Animal Science 83, 147152.
Naumann, K. & Bassler, R. (1997). VDLUFA-Methodenbuch Band III. Die chemische Untersuchung von Futtermitteln. 4. Ergänzungslieferung. Darmstadt, Germany: VDLUFA-Verlag.
Ohshima, M. & McDonald, P. (1978). A review of the changes in nitrogenous compounds of herbage during ensilage. Journal of the Science of Food and Agriculture 29, 497505.
Owens, V. N., Albrecht, K. A. & Muck, R. E. (1999 a). Protein degradation and ensiling characteristics of red clover and alfalfa wilted under varying levels of shade. Canadian Journal of Plant Science 79, 209222.
Owens, V. N., Albrecht, K. A., Muck, R. E. & Duke, S. H. (1999 b). Protein degradation and fermentation characteristics of red clover and alfalfa silage harvested with varying levels of total nonstructural carbohydrates. Crop Science 39, 18731880.
Papadopoulos, Y. A. & McKersie, B. D. (1983). A comparison of protein degradation during wilting and ensiling of six forage species. Canadian Journal of Plant Science 63, 903912.
Parveen, I., Threadgill, M. D., Moorby, J. M. & Winters, A. (2010). Oxidative phenols in forage crops containing polyphenol oxidase enzymes. Journal of Agricultural and Food Chemistry 58, 13711382.
Pichard, G. R., Tesser, B. R., Vives, C., Solari, C., Hott, A. & Larraín, R. E. (2006). Proteolysis and characterization of peptidases in forage plants. Agronomy Journal 98, 13921399.
Seguin, P. & Mustafa, A. F. (2003). Chemical composition and ruminal nutrient degradabilities of fresh and ensiled kura clover (Trifolium ambiguum M.B.). Canadian Journal of Animal Science 83, 577582.
Seng, M., Bonorden, S., Nissen, J., Isselstein, J. & Abel, H. (2008). Fermentation patterns and nutrient contents of forb-containing silages and their effects on microbial fermentation in the artificial rumen system RUSITEC. Journal of Agricultural Science, Cambridge 146, 333341.
Seyfarth, W., Knabe, O. & Weise, G. (1989). Protein degradation during the silaging of green fodder. Archiv fuer Tierernaehrung 39, 685691.
Sniffen, C. J., O'Connor, J. D., Van Soest, P. J., Fox, D. G. & Russell, J. B. (1992). A net carbohydrate and protein system for evaluating cattle diets: II. Carbohydrate and protein availability. Journal of Animal Science 70, 35623577.
Sullivan, M. L. & Hatfield, R. D. (2006). Polyphenol oxidase and o-diphenols inhibit postharvest proteolysis in red clover and alfalfa. Crop Science 46, 662670.
Tremblay, G. F., Bélanger, G., McRae, K. B. & Michaud, R. (2001). Proteolysis in alfalfa silages made from different cultivars. Canadian Journal of Plant Science 81, 685692.
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. Journal of Dairy Science 74, 35833597.
Verbič, J., Ørskov, E. R., Žgajnar, J., Chen, X. B. & Žnidaršič-Pongrac, V. (1999). The effect of method of forage preservation on the protein degradability and microbial protein synthesis in the rumen. Animal Feed Science and Technology 82, 195212.
Voss, N. (1967). Investigations on protein decomposition in grass and lucerne silage. Das wirtschaftseigene Futter 13, 130145.
Weinberg, Z. G. & Ashbell, G. (2003). Engineering aspects of ensiling. Biochemical Engineering Journal 13, 181188.
Weissbach, F. & Strubelt, C. (2008). Die Korrektur des Trockensubstanzgehaltes von Silagen als Substrat für Biogasanlagen. In Kurzfassungen der Referate, 120. VDLUFA-Kongress, pp. 2931. Jena, Germany: VDLUFA-Verlag.
Winters, A. L., Minchin, F. R., Michaelson-Yeates, T. P. T., Lee, M. R. F. & Morris, P. (2008). Latent and active polyphenol oxidase (PPO) in red clover (Trifolium pratense) and use of a low PPO mutant to study the role of PPO in proteolysis reduction. Journal of Agricultural and Food Chemistry 56, 28172824.

The effect of cultivar on the changes in protein quality during wilting and ensiling of red clover (Trifolium pratense L.)

  • M. KRAWUTSCHKE (a1), N. WEIHER (a1), J. THAYSEN (a2), R. LOGES (a1), F. TAUBE (a1) and M. GIERUS (a1)...


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed