Effects of plant species, stage of maturity, and level of formic acid addition on lipolysis, lipid content, and fatty acid composition during ensiling

Forage type and management influences the nutritional quality and fatty acid composition of ruminant milk. Replacing grass silage with red clover (RC; Trifolium pratense L.) silage increases milk fat 18:3n-3 concentration. Red clover has a higher polyphenol oxidase (PPO) activity compared with grasses, which has been suggested to decrease lipolysis in silo and in vivo. The present study characterized the abundance and fatty acid composition of esterified lipid and NEFA before and after ensiling of grass and RC to investigate the influence of forage species, growth stage, and extent of fermentation in silo on lipolysis. A randomized block design with a 2 x 3 x 4 factorial arrangement of treatments was used. Treatments comprised RC or a mixture of timothy (Phleum pratense L.) and meadow fescue (Festuca pratensis Huds.) harvested at 3 growth stages and treated with 4 levels of formic acid (0, 2, 4, and 6 L/t). Lipid in silages treated with 0 or 6 L/t formic acid were extracted and separated into 4 fractions by TLC. Total PPO activity in fresh herbage and the content of soluble bound phenols in all silages were determined. Concentrations of 18:3n-3 and total fatty acids (TFA) were higher (P < 0.001) for RC than for grass. For both forage species, 18:3n-3 and TFA content decreased linearly (P < 0.001) with advancing growth stage, with the highest abundance at the vegetative stage. Most of lipid in fresh RC and grass herbage (97%) was esterified, whereas NEFA accounted for 71% of TFA in both silages. Ensiling resulted in marginal increases in TFA content and the amounts of individual fatty acids compared with fresh herbages. Herbage total PPO activity was higher (P < 0.001) for RC than grass (11 vs. 0.11 mu katal/g leaf fresh weight). Net lipolysis during ensiling was extensive for both forage species (660 to 759 g/kg fatty acid for grass and 563 to 737 g/kg fatty acid for RC). Formic acid application (0 vs. 6 L/t) resulted in a marked decrease (P = 0.026) in net lipolysis during the ensiling of RC, whereas the opposite was true (P = 0.026) for grass. In conclusion, results suggest that formic acid addition during the ensilage of RC decreases lipolysis in silo. For both plant species, total PPO activity was not associated with the extent of lipolysis in silo. However, bound phenols formed via PPO activity appear to have a role in protecting lipid and protein against degradation in grass and lowering proteolysis of RC during ensiling.