Humphreys, M. O., Feuerstein, U., Vandewalle, M., Baert, J. (2010). Ryegrasses, In: Handbook of Plant Breeding: Fodder Crops and Amenity Grasses, Volume 5, Springer New York, pp. 211-260, 2010In the temperate regions of the world ryegrasses comprise the main sown forage grasses of which the outbreeding perennial, Italian and Westerwolths ryegrasses are the most economically important. This chapter presents information on the origin and systematics of these forage grasses which share a high degree of genome ancestry with the cereals originating in the eastern Mediterranean basin. It considers how plant breeding has enhanced natural genetic resources to produce valuable new varieties with improved production and livestock nutrition characteristics together with increased tolerance of environmental stresses caused by biotic (pests and diseases) and abiotic (temperature, water and mineral extremes) pressures. The challenge of maintaining the optimum balance between vegetative growth and seed production is also addressed. Breeding methodologies based on traditional techniques are discussed and the integration of new biotechnologies into breeding programmes is reviewed. Breeding achievements are evaluated and future goals considered in relation to the increasingly diverse demands placed on grassland to provide ecosystem services, amenity value and systematic breeding as well as providing feed for ruminants

The fatty acid (FA) concentration of herbage and lipid metabolism in silage, mainly oxidation and lipolysis, of different species (perennial ryegrass, red clover and white clover) and three cultivars of white and red clover at three cutting dates in the growing season (April, July and October) were studied. FA concentration and composition was strongly affected by species and cutting date. Perennial ryegrass had lower concentrations of C16:1, C18:0, C18:1 and C18:2 than red and white clover. Within red and white clover, the effect of cultivar was small. Oxidation of C18:3 during wilting was different between species and cutting date despite similar wilting conditions. Lipolysis in silage was also influenced by cutting date, species and to some extent by cultivar. Furthermore, in some cuts silages of red and white clover displayed a lower lipolysis than silage of perennial ryegrass. On average, over the three cutting dates proportionately 0.903, 0.864 and 0.857 of the membrane lipids in perennial ryegrass, red clover and white clover were hydrolysed during ensiling. In red clover this could be due to the lipid-protecting properties of polyphenol oxidase (PPO) activity. This was not observed in perennial ryegrass or white clover. Nevertheless, differences in lipolysis in silage between cultivars of red clover were not correlated with PPO activity

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.

The goal of this paper was, first, to study the effect of red clover polyphenol oxidase (PPO) activity on protein-bound phenols and measured lipase activity in vitro and, second, to study the effect of PPO activation, measured as an increase in protein-bound phenols, as a result of degrees of damaging (not damaged, crushed, and freeze/thawed) of red clover before wilting on measured enzyme activity and in vitro lipid metabolism when incubated in a phosphate buffer. There was a positive relation between PPO activity and the occurrence of protein-bound phenols with a concomitant decrease in measured lipase activity, indicating a possibility to a direct inhibition of enzymes as a result of protein-bound phenols. Furthermore, damaging can activate PPO in red clover, measured as an increase in protein-bound phenols during wilting [0.7-20.6 nmol of tyrosine equiv (mg of protein)(-1)], again with a concomitant decrease in measured lipase activity [41.3-20.3 mumol of p-nitrophenyl butyrate (PNPB) min(-1) (mg of protein)(-1)]. Lipid metabolism during incubation of these forages in a phosphate buffer with ascorbic acid was only influenced by damaging when wilted for 24 h, with a lower lipolysis in crushed and freeze/thawed (52.9 and 32.6%, respectively, after 8 h of incubation) material compared to all other treatments (on average 60.4% after 8 h of incubation).

Most often, farmers consider red clover an unattractive forage because of its low ensilability. Nevertheless, several in vivo and in vitro experiments also showed advantages of red clover silages such as decreased rumen biohydrogenation of polyunsaturated fatty acids. This has been attributed to a possible protective role of protein-bound phenols, with polyphenol oxidase playing a key role in their formation. This enzyme is active in red clover, but not in other green forages, such as, for example, perennial ryegrass. Therefore, the aim was to study the lipid metabolism within red clover/ryegrass mixtures in lab scale silages and during in vitro rumen batch incubations. Ensilability of red clover increased with higher proportions of ryegrass in the silage mixture. However, the lipid-protecting mechanism of red clover does not seem to occur in the co-ensiled ryegrass as lipolysis of polar lipids linearly increased with increasing proportions of ryegrass (86.0%, 91.6%, 89.9%, 93.1% and 95.6% in 60-day-old silages with 100/0, 75/25, 50/50, 25/75 and 0/100 red clover/ryegrass, respectively). Rumen lipolysis and biohydrogenation of C18:3n-3 and C18:2n-6 were negatively related to red clover proportions in the silage mixtures. The lipid-protective mechanism in red clover silages is confirmed, but it seems not to be transferred to lipids in co-ensiled forages.Keywords: lipid metabolism, red clover, in vitro rumen, silage ImplicationsLipids in red clover silages are less degraded as compared with lipids in ryegrass silages, which have been attributed to a lipid-protective mechanism in red clover. This implicates that less oilseeds would be required to reach milk with a healthier fatty acid profile when feeding red clover instead of ryegrass silages. However, the lipid protection is not transferred to grass lipids in mixed grass-red clover silages.