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Title:   Author  item
                                            LEE, MICHAEL - INST. OF GRASSLAND & ENV.item
                                            MINCHIN, FRANK - INST. OF GRASSLAND & ENV.item
                                            Hatfield, Ronalditem
                                            Sullivan, Michael
  Submitted to: Symposium Proceedings
                                    Publication Type: Proceedings
                                    Publication Acceptance Date: 2/15/2007
                                    Publication  Date: 4/2/2007
                                    Citation: Lee, M.R., Minchin, F.R., Hatfield, R.D., Sullivan, M.L. 2007. Red clover polyphenol oxidase reduces ruminal lipolysis in in vitro batch culture. In: Proceedings of the British Society of Animal Science. April 2-4, 2007, Southport, United Kingdom. p. 25.Interpretive Summary:
                                Technical Abstract: Introduction. It has been shown that the rate of lipolysis and proteolysis differs significantly between red clover genotypes with different levels of polyphenol oxidase (PPO) activity (Lee et al. 2004). Sullivan and Hatfield, (2006) reported the development of genetically modified isolines of red clover with the PPO1 gene silenced. This material was used to examine the role of the red clover PPO enzyme on lipolysis and ultimately C18 polyunsaturated fatty acid biohydrogenation in batch culture. If the role of PPO in reducing ruminal lipolysis of plant lipids is proven it would influence breeding strategies for forages which exhibit this trait in an attempt to increase the levels of beneficial PUFA and decrease detrimental trans and saturated fatty acids in animal products. 

Materials and methods. Anaerobic incubation medium was prepared as described by Goering and Van Soest (1970) and 20ml dispensed under CO2 into 24 flasks maintained at 39oC. Fresh red clover from silenced PPO1 gene plants (S) and isogenic active PPO1 gene plants (A) were cut 3 cm above soil level. The tissue was crushed and cut into 5 mm strips, with a sample retained at -20oC to measure PPO activity. Four grams of fresh material was loaded into each incubation flask. Three bottles were allocated to each time point (0, 1, 6 and 24 h) for each treatment (S and A). The flasks were then inoculated with 10 ml of strained rumen liquor (from two rumen fistulated cows and strained through a double layer of muslin). The flasks were sealed and incubated at 39oC in the dark with continuous CO2 purging. At each time point the appropriate incubation flasks were removed and 40 ml of isopropanol : chloroform (1:1 v/v) along with 1 ml of internal standard (2.5 mg C23:0 / ml chloroform) added and the lipid extracted and fractionated by TLC as described by Lee et al. (2004). Lipolysis was calculated by expressing the decrease in the proportion of membrane lipid between the initial time point T0 and incubation time point Tx, and then analysed using a repeated measures analysis of variance (Genstat 8.1, Lawes, Agricultural Trust, 2005). 

Results. PPO activity for the PPO1-silenced red clover (S) and the active PPO1 red clover (A) were 0 and 11.4 nkat/mg protein. Fig. 1. shows the extent of lipolysis of S and A. At all time points lipolysis in the S treatments were higher than the A treatments with 6 and 24 h being significantly higher (P<0.001) than S.

Conclusions. The PPO1-silenced red clover had significantly higher lipolytic activity in batch culture than the red clover with the active PPO1 gene. This result, obtained with isogenic lines, provides strong evidence for a role of PPO in reducing the extent of lipolysis in the presence of rumen micro-organisms. Mechanistically this may be due to a binding of quinones to the lipid or/and the formation of protected protein/lipid complexes


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