Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation

Abstract

This paper describes the relationship between protein-bound phenols in red clover, induced by different degrees of damaging before wilting and varying wilting duration, and in silo lipid metabolism. The ultimate effect of these changes on rumen biohydrogenation is the second focus of this paper. For this experiment, red clover, damaged to different degrees (not damaged (ND), crushing or frozen/thawing (FT)) before wilting (4 or 24 h) was ensiled. Different degrees of damaging and wilting duration lead to differences in polyphenol oxidase (PPO) activity, measured as increase in protein-bound phenols. Treatment effects on fatty acid (FA) content and composition, lipid fractions (free FAs, membrane lipids (ML) and neutral fraction) and lipolysis were further studied in the silage. In FT, red clover lipolysis was markedly lower in the first days after ensiling, but this largely disappeared after 60 days of ensiling, regardless of wilting duration. This suggests an inhibition of plant lipases in FT silages. After 60 days of ensiling no differences in lipid fractions could be found between any of the treatments and differences in lipolysis were caused by reduced FA proportions in ML of wilted FT red clover. Fresh, wilted (24 h) after damaging (ND or FT) and ensiled (4 or 60 days; wilted 24 h; ND or FT) red clover were also incubated in rumen fluid to study the biohydrogenation of C18:3n-3 and C18:2n-6 in vitro. Silages (both 60 days and to a lower degree 4 days) showed a lower biohydrogenation compared with fresh and wilted forages, regardless of damaging. This suggests that lipids in ensiled red clover were more protected, but this protection was not enhanced by a higher amount of protein-bound phenols in wilted FT compared with ND red clover. The reduction of rumen microbial biohydrogenation with duration of red clover ensiling seems in contrast to what is expected, namely a higher biohydrogenation when a higher amount of FFA is present. This merits further investigation in relation to strategies to activate PPO toward the embedding of lipids in phenol-protein complexes.Keywords: polyphenol oxidase, fatty acid, PUFA, biohydrogenation, lipolysis ImplicationsRed clover polyphenol oxidase (PPO) has the potential to protect lipids against lipolysis in silage and lipolysis and biohydrogenation in the rumen, leading to higher concentrations of polyunsaturated fatty acids in ruminant meat and milk. PPO is a stress-activated enzyme, which is active in red clover. The goal of this paper was to study the effect of PPO activity due to damaging after mowing, measured as an increase in protein-bound phenols, and wilting duration on lipid metabolism in the silage, mainly lipolysis, and lipolysis and biohydrogenation in the rumen.

Red clover polyphenol oxidase and lipid metabolism

“…Relative lipolysis in the study by Van Ranst et al (2010) was lowest in the first days after ensiling (0.652, 0.355 and 0.750 for FT 4 h wilted, FT 24 h wilted and high v. low PPO activity, respectively), whereas the difference in the control largely disappeared after 60 to 90 days of ensiling (0.905, 0.921 and 0.929 for FT 4 h wilted, FT 24 h wilted and high v. low PPO activity, respectively). It can be assumed that during the early stages of ensiling, when silage fermentation is still at a low rate, the vast majority of lipolysis will be mediated through plant lipases.…”

“…All the results indicate a biohydrogenation inhibiting role of PPO, as after 24 h incubation (or continuous culture incubations in the study by Loor et al (2003)), biohydrogenation was consistently lower for red clover with a high v. a low PPO activity or red clover compared with cocksfoot. Damaging red clover by means of FT and ensiling also resulted in a decrease in ruminal biohydrogenation after 24 h incubation (Van Ranst et al, 2010), indicating that damaging and ensiling red clover could also result in the protection of plant lipids.…”

“…In silo studies In Figure 4, it is demonstrated using data of two studies (Lee et al, 2008b;Van Ranst et al, 2010) that in silo lipolysis of glycerol-based cell and organelle membrane lipids can be reduced when high PPO activity or activated PPO is present. In Van Ranst et al (2010), the control silage was undamaged; red clover wilted 4 h before ensiling.…”

“…In Van Ranst et al (2010), the control silage was undamaged; red clover wilted 4 h before ensiling. In this treatment, the lack of increase in protein-bound phenols after wilting suggests that PPO activation was only minor.…”

“…A, B and C represent undamaged, crushed or frozen/thawed red clover, respectively, after 24 h wilting incubated in a buffer with ascorbic acid with fresh red clover as a control; D (Van Ranst, 2009) and E (Lee et al, 2004) are red clover mixed in a buffer without ascorbic acid, with ryegrass and low polyphenol oxidase red clover as a control, respectively; F (Lee et al, 2007) and G (Lee et al, 2006b) correspond with red clover and cocksfoot without ascorbic acid compared with ascorbic acid as a control. Figure 4 Relative lipolysis during ensiling of red clover; A represents lipolysis as a mean of three treatments, which did not significantly differ red clover crushed and wilted for 4 h and crushed or undamaged and wilted for 24 h (A); red clover frozen/thawed and wilted for 4 (B) or 24 h (C), expressed relative to not damaged 4 h wilted red clover (Van Ranst et al, 2010). D represents lipolysis in silage of red clover with normal Polyphenol oxidase (PPO) activity, expressed relative lipolysis in red clover with very low PPO activity (Lee et al, 2008b).…”