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Title:   Author  item
                                            LEE, MICHAEL - INST GRASSLAND ENVIR RESitem
                                            TWEED, J - INST GRASSLAND ENVIR RESitem
                                            SCOLLAN, N - INST GRASSLAND ENVIR RESitem
                                            Sullivan, Michael
  Submitted to: Symposium Proceedings
                                    Publication Type: Proceedings
                                    Publication Acceptance Date: 12/11/2007
                                    Publication  Date: 3/31/2008
                                    Citation: Lee, M.R., Tweed, J.K., Scollan, N.D., Sullivan, M.L. 2008. Rumen microbe adaptation to red clover polyphenol oxidase protein and lipid protection. In: Proceedings of the British Society of Animal Science, March 31-April 2, 2008, Scarborough, United Kingdom. p. 30.Interpretive Summary:
                                Technical Abstract: Introduction: Polyphenol oxidase (PPO) has been shown to reduce both proteolysis and lipolysis in incubated red clover (Lee et al. 2004). However it has not been determined whether rumen microbes can adapt to utilize PPO-protected protein and lipid. This study investigated whether rumen inoculum from cows which were offered red clover silage resulted in higher levels of proteolysis and lipolysis in red clover (+PPO) and also in red clover with the PPO1 gene silenced (–PPO), than rumen inoculum from cows which were offered grass silage, due to microbial adaptation.

Materials and Methods: PPO1 gene silenced (PPO-) and wild type (PPO+) plants grown in controlled conditions were harvested at 5 cm above soil level, crushed, and cut into 5-mm strips and wilted for 1 h. Two rumen-fistulated cows were offered ad libitum either red clover silage (RC) or grass silage (G) for two 2-week periods in a 2 x 2 Latin square. One liter of hand-squeezed rumen inoculum was collected from each cow at the end of each period and transferred back to the laboratory in a temperature-regulated flask (39 degrees C). At the end of the first period, the cows were given 2 weeks rest before starting the alternative silage in the second period. Red clover (2.5 g) was weighed into 32 tubes, 16 PPO+ and 16 PPO- (for each period) which provided duplicate tubes at each time point (0, 2, 6, and 24 h). Into each tube, 7.5 ml of anaerobic buffer, 0.35 ml reducing agent, and 2.5 ml of either RC or G strained rumen inoculum were added before being purged with carbon dioxide and incubated at 39 degrees C. At each time point, the supernatant was sub-sampled for free amino acid analyses. The tubes were then frozen with liquid nitrogen and freeze-dried. The lipid was extracted with 3 X 5 ml of chloroform:methanol (2:1; v/v) and dried down under nitrogen at 50 degrees C. The sample was then fractionated by thin-layer chromatography, and the lipid fractions were bimethylated before being run on GC. Lipolysis was calculated as the proportional loss of membrane lipid, and rise in free amino acids was used as a predictor of proteolysis.  

Results: Data are presented on lipolysis and the rise in free amino acids (proteolysis), respectively, of the two red clovers + and – PPO in either RC or G silage fed rumen inoculum. In both rumen inoculums, the levels of lipolysis and proteolysis were lower (P<0.05) in the PPO+ treatment than the PPO- treatment over time. RC inoculum did not result in a higher level of proteolysis or lipolysis than the G inoculum. 

Conclusions: Lipolysis and proteolysis in PPO+ were significantly lower than PPO- in both inoculum regimes, with no difference between RC or G inoculum. The results suggest that rumen-micro-organisms grown in an environment containing PPO-protected phenol-bound protein do not adapt to increase the utilization of the protected protein to any great extent


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