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J Dairy Sci. 2018 Oct;101(10):8954-8964. doi: 10.3168/jds.2018-14763. Epub 2018 Jul 19.

Effects of mixing red clover with alfalfa at different ratios on dynamics of proteolysis and protease activities during ensiling.

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Institute of Grassland Science, China Agricultural University, Beijing, China, 100193.
Institute of Livestock Science, Jiangsu Academy of Agricultural Science, Nanjing, China 210014.
College of Forestry and Landscape Architecture, South China Agricultural University, Guangzhou, China 510642.
Department of Animal Sciences, University of Florida, Gainesville 32611.
Institute of Grassland Science, China Agricultural University, Beijing, China, 100193. Electronic address:


This study was conducted to study the effects of ensiled alfalfa (Medicago sativa) and red clover (Trifolium pratense) at different ratios on dynamics of fermentation parameters, N distribution, protein fractions, and protease activities during ensiling. Alfalfa and red clover were harvested and wilted to 35 and 25% dry matter, respectively, chopped to 1 cm, mixed, weighed into 1.0-L buckets at a density of 700 g/L, and ensiled for 1, 3, 7, 15, and 30 d at 30°C. The treatments were mixing ratio of alfalfa to red clover at 100:0, 70:30, 50:50, 30:70, and 0:100 (R0, R30, R50, R70, and R100, respectively; fresh weight). For each ensiling duration, 3 replicates of each treatment were prepared. With increasing proportion of red clover in silage, total N content and proportions of nonprotein N, peptide N, free amino acid N, and NH3-N decreased linearly, and PC (indigestible true protein, acid detergent insoluble N) proportion increased linearly after ensiling. Moreover, the final pH was lower in R50 and R100 than R0 (4.29, 4.20 vs. 4.48, respectively) on d 30. Also, lactic acid concentration on d 30 was higher in R50, R70, and R100 silage compared with R0 (7.77, 7.66, and 8.76 vs. 6.34, % of dry matter, respectively). The proportion of NH3-N in R50 was lower than in R0 but closer to R100 after ensiling. During ensiling, proteases including carboxypeptidase, aminopeptidase, and acid proteinase activities decreased as red clover proportion increased. However, no differences were detected in aminopeptidase and acid proteinase activities among R50, R70, and R100 during ensiling. Overall, 50:50 was the optimal mixing ratio of alfalfa with red clover, showing good fermentation quality with lower pH and higher lactic acid concentration, reduced protease activities and proteolysis compared with pure alfalfa silage, and also more total N content than pure red clover silage.


alfalfa; proteolysis; red clover; silage

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