Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen fluid

This experiment aims to determine whether polyphenol oxidase (PPO) can reduce the extent of lipolysis and the consequent polyunsaturated fatty acid loss through microbial biohydrogenation in red clover when incubated in the presence of rumen fluid. PPO is involved in the browning reaction of red clover leaves when cut or crushed and exposed to air. It starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins and have been shown to reduce proteolysis and lipolysis in silo. Two lines of red clover (cv. Milvus), a genotypic mutant with reduced PPO activity (L) and the wild type (H) with a high level of PPO activity, were cut 3 cm above soil level, crushed and cut into 1cm strips before being loaded into incubation bottles. These were then incubated in anaerobic buffer at 39 ◦C in either the absence (−) or the presence (+) of rumen microorganisms. The incubations were then compared over a 24 h time course in terms of lipolytic activity. Characterization of the tissues showed PPO activities of 25.3 and 5.13 U g−1 fresh weight for H and L, respectively. Lipolysis, measured as the proportional decline in the membrane lipid, was reduced (P <0.001) with increasing PPO activity in both the presence (+) and absence (−) of rumen microorganisms. However, values were significantly higher (P <0.001) in the presence of rumen microorganisms, with values after the 24 h incubation of 0.28, 0.42, 0.72 and 0.82 for H−, L−, H+ and L+, respectively. Biohydrogenation of C18:2 and C18:3 polyunsaturated fatty acids were significantly lower in the H+ treatment than the L+ treatment, with mean values after 24 h incubation of 53% and 57% (P <0.05) for C18:2 and 65% and 74% (P <0.01) for C18:3, respectively. Changes that occurred in the lipid fractions (membrane lipid, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce polyunsaturated fatty acid loses in the rumen.